ε-caprolactam

Endpoint summary

Description of key information

Based on the available data from a study compareable to OECD Guideline 408 the NOEAL for general systemic effects in mal rats was 29 mg/kg bw/d and 342 mg/kg bw/d in female rats. The NOAEC in rats for systemic inhalation effects was 0.245 mg/l and for local effects 0.066mg/l based on the results of a study performed described by TSCA, EPA September 195 and May 1989.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no information on the analytical examination of doses, ophthalmoscopic and neurobehavioural examination not performed

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Broekman Institute, Stiphout, The Netherlands
- Mean weight at study initiation: male 42.6 g, female 41.2 g
- Housing: 5 per cage
- Diet (e.g. ad libitum): test compound was thoroughly mixed into the stock diet
- Water (e.g. ad libitum): tap water

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
- The following percentage composition of stock diet was used:
yellow maize 29.5, brewer's yeast 3, whole wheat 36, grass meal 3, soyabean-oil meal 10, soyabean oil 3, meat scraps 4, vitamin preparations 0.5, fish meal 8, trace mineralized salt 0.5, dried whey 2, and calcium phosphate 0.5. The test compound was thoroughly mixed into the stock diet by means of a modified meat cutter (Stefan).
- Rate of preparation of diet (frequency): The diets were freshly prepared once a fortnight

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously

Dose / conc.:

0.05 other: % (nominal in diet)

Remarks:

29 mg/kg bw in males and 35 mg/kg bw in females (actual ingested)

Dose / conc.:

0.1 other: % (nominal in diet)

Remarks:

60 mg/kg bw in males and 70 mg/kg bw in females (actual ingested)

Dose / conc.:

0.25 other: % (nominal in diet)

Remarks:

146 mg/kg bw in males and 170 mg/kg bw in females (actual ingested)

Dose / conc.:

0.5 other: % (nominal in diet)

Remarks:

296 mg/kg bw in males and 342 mg/kg bw in females (actual ingested)

Dose / conc.:

1 other: % (nominal in diet)

Remarks:

592 mg/kg bw in males and 704 mg/kg bw in females (actual ingested)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The toxicity of caprolactam (CAP) was examined in a range-finding test with weanling albino rats fed the test substance at dietary levels of 0, 0.2, 1 and 5 % for 28 days. CAP at levels of 5 % and 1 % in the diet caused growth depression and increased liver and kidney weights. Histopathological changes in liver and kidney were observed in the 5 % group in both sexes. In the 1 % group slight histopathological changes were observed only in the kidneys of males. On the basis of the present results 1% CAP is expected to be an effect level in a sub-chronic (90-day) toxicity study.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 13 week
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked: haemoglobin, packed cell volume, red blood cell counts and total and differential white blood cell counts


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of experiment
- Animals fasted: No data
- How many animals: all
- Parameters checked: glutamic-pyruvic transaminase (SGPT), glutamic-oxalacetic transaminase (SGOT), alkaline phosphatase (SAP), total serum
protein, albumin and albumin-globulin ratio


URINALYSIS: Yes
- Time schedule for collection of urine: 13 week
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: appearance, pH, glucose, albumin, occult blood, ketones and microscopy of the sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

The following organs were weighed: heart, kidneys, spleen, liver, brain, testicles/ovaries, thymus, pituitary, thyroids and adrenals

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities of condition or behaviour were observed.

Mortality:

no mortality observed

Description (incidence):

During the course of the experiment no deaths occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight growth depression occurred at the 1.0 % feeding level in both sexes. This growth depession was only biologically relevant in males (15 and 11% at week 2 and 4).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Decreased food consumption was observed at the highest feeding level in both sexes during week 2, 3 and 4. At 0.5 % food consumption was relatively low in week 2 and 3 in both sexes. Towards the end of the experiment food consumption was comparable in all groups.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency in males at the highest feeding level was slightly lower than in the other test groups. The food efficiency figure obtained in control males was low, probably as a result of relatively low body weights at week 4 which had disappeared not until week 10.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological data showed no distinct differences between groups. Total leucocytes count was increased in males of some experimental groups, but there was no trend towards higher average leucocytes counts with an increase of Caprolactam in the diet.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant differences occurred in serum enzyme values amongst the various groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The results of the urine analyses showed no changes in the composition of the urine which could be attributed to the feeding of Caprolactam.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative weight of the kidneys and testicles were increased (25 and 18%) at 1.0 % in males. The relative weight of the liver was increased in both sexes (19% in males and 14% in females) at 1.0 %. All other organ weights of test groups were comparable with the control values. The mathematically significantly decreased heart weight of females at 0.5 % is not considered of toxicological importance, because it was not observed at the 1.0 % level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At autopsy, the kidneys of male rats showed a pale-greenish discolouration at the highest dose level. Other gross changes attributable to the ingestion of Caprolactam were not seen.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At microscopic examination pathological changes related to the ingestion of Caprolactam were found in the kidneys of males only. These changes consisted of hyaline-droplet degeneration of the proximal convoluted tubules in the kidneys of nearly all male rats at 0.1 % Caprolactam and above. Its degree was distinctly dose-related. The swollen epithelial cells of these tubules showed a granular cytoplasm,caused by large accumulations of tiny droplets of precipitated proteinaceous material. The droplets stained deeply red with Azan. At the 0.05 % level these changes were completely absent.
The other abnormalities were about equally distributed amongst test and control animals. The pathological examinations indicate that the ingestion of Caprolactam at levels of 0.1 % and above is nephrotoxic for male rats of this particular strain (SD).

Dose descriptor:

NOAEL

Effect level:

29 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: hyaline-droplet degeneration of the proximal convoluted tubules in the kidneys

Dose descriptor:

NOEL

Effect level:

342 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: liver weight

Critical effects observed:

not specified

Conclusions:

Based on hyaline-droplet degeneration of the proximal convoluted tubules in the kidneys, the NOAEL for males was found to be 29 mg/kg bw/day.
In females, the NOAEL was 342 mg/kg bw based on increase in relative liver weights.
The findings in male kidneys are supposed to be of no relevance for other species including humans (Haschek and Rousseaux, Fundamentals of Toxicologic Pathology, Academic Press, 1996). Therefore, CAP does not require any classification.

Executive summary:

A study similar to the OECD Guideline 408 was performed. A distinct gender variance in systemic toxicity of CAP was observed in this 90 day continuous feeding study with Sprague-Dawley rats.

Various dietary levels up to 1.0 % resulted in distinct ill effects as shown by growth depression, decreased food intake and increased weights of the liver at 1.0 % in both sexes.

In males discoloration of the kidneys and increased relative weights of kidneys and testicles at 1.0 % and microscopically changes of the kidneys at 0.1 % and above was observed. The kidneys of male rats are more susceptible to Caprolactam than those of females. Possibly a relationship exists between the presence of this type of degeneration in males only and the well-known higher urinary protein excretion in male rats than in females (Sellers et al. Am. J. Physiol. 163; 662-667, 1950).

Based on hyaline-droplet degeneration of the proximal convoluted tubules in the kidneys, the NOAEL for males was found to be 29 mg/kg bw/day.

In females, the NOAEL was 342 mg/kg bw based on increase in relative liver weights.

The findings in male kidneys are supposed to be of no relevance for other species including humans (Haschek and Rousseaux, Fundamentals of Toxicologic Pathology, Academic Press, 1996). Therefore, CAP does not require any classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

29 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: described by TSCA, EPA September 1985 and May 1989

GLP compliance:

yes

Remarks:

(Huntington Life Science)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Caprolactam
- Physical state: white crystalline solid
- Analytical purity: 99.9%
- Stability under test conditions: stable for study duration
- Storage condition of test material: room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6 weeks
- Mean weight at study initiation: males 178 g (151-205 g), females 143 g (110-167 g)
- Housing: 2 per cage
- Diet (e.g. ad libitum): Certified Rodent Diet, No. 5002, PMI feeds Inc, MO.
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-27
- Humidity (%): 21-74
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

water

Remarks on MMAD:

MMAD / GSD: 2.9 µm/1.7

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: The placement of the animal in the whole body exposure chamber was rotated at each exposure to ensure uniform exposure of the animals.
- System of generating particulates/aerosols: A 1:1 solution, prepared by dissolving Caprolactam in an equal amount, by weight, of distilled demineralized water, was placed in a 250 ml Erlenmeyer flask and continuously stirred on a stirplate. The solution was fed directly, via an FMI Fluid Metering Pump, into the fluid inlet of a Spraying System, Inc. air atomizing nozzle (JCO-SS with a lA spray setup). The aerosol so generated was mixed with incoming house air by turbulence. Conditioned house air was metered into the control chamber.
- Temperature, humidity in air chamber: 21-32°C, 28-65%
- Method of particle size determination: Particle size distribution samples were withdrawn once during each exposure and determined using an aerodynamic particle sizer (TSI Aerodynamic Particle Sizer, Model 3300, and a Model 3302 TSI Aerosol Diluter). The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated.


TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, chamber concentration determinations were performed six times daily using gravimetric sampling. In addition, for each daily exposure group, one sample was also analyzed by high-pressure liquid chromatography (HPLC) using a Hewlett-Packard 1050 LC system and a UV detector.
- Samples taken from breathing zone: yes


VEHICLE (if applicable)
- Concentration of test material in vehicle: 1:1 aqueous solution with Caprolactam
- Lot/batch no. of vehicle (if required): Prepared by Pharmacy from water supplied by the Elizabethtown Water Company
- Purity of vehicle: Monthly analysis was provided by the Elizabethtown Water Company

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean (±SD) analytical exposure concentrations were 0±0, 23±4.9, 66±20 and 244±37 mg/m3 (approx. 0±0, 0.023±0.0049, 0.066±0.02 and 0.245 ±0.037 mg/l).
Gravimetrically determined exposure concentrations were (mean ±SD) 0.063 ±0.19, 24±5.6, 70±18 and 243±36 mg/m3 for the 13 weeks of exposure.

Duration of treatment / exposure:

13 weeks plus 4 week-recovery

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0 mg/L air (nominal)

Remarks:

0 mg/mL analytical conc.

Dose / conc.:

0.025 mg/L air (nominal)

Remarks:

0.023 mg/mL analytical concentration

Dose / conc.:

0.075 mg/L air (nominal)

Remarks:

0.066 mg/mL analytical concentration

Dose / conc.:

0.25 mg/L air (nominal)

Remarks:

0.245 mg/mL analytical concentration

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Post-exposure recovery period in satellite groups: An additional 10 animals/sex/group were similarly exposed and then held for a 4-week recovery period

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study and at least once during each exposure
- Cage side observations checked: mortality, general appearance and signs of toxic or pharmacological effects


BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly during study period and prior to the terminal and recovery sacrifices


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the first exposure and prior to the terminal sacrifice
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the exposure and recovery terminations
- Anaesthetic used for blood collection: Yes (light carbon dioxide anesthesia)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: erythrocyte count and morphology, total and differential leukocyte count, platelet count, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, prothrombin time, and activated partial thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the exposure and recovery terminations
- Animals fasted: Yes
- How many animals: all
- Parameters checked: serum alanine aminotransferase, serum aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, fasting glucose, total serum protein, albumin, globulin (calculated as total protein - albumin = globulin), albumin/globulin ratio (calculated), total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, and y-glutamyl transferase


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pretest and Weeks 5, 9, and 14 and Recovery (Week 19)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other: For homecage observations, each rat was observed and scored for posture, vocalization, and palpebral closure. On removal from the cage, each animal was observed and scored for ease of removal, ease of handling, lacrimation, appearance of fur, and salivation. Open-field evaluations included observations and scoring of gait, degree of locomotion, arousal, abnormal movements including tremors or convulsions, exophthalmia, piloerection, and presence or absence of diarrhea and urination. Reflex assessments consisted of scoring of responses to approach, tail pinch, finger snap, pupillary response, and air righting reflex. Additionally, landing foot splay and fore- and hindlimb grip strengths were assessed. Motor activity was monitored using an automated Photobeam Activity System (San Diego Instruments, Inc.). Sessions were 60 min in length; each session was divided into 12 intervals of 5-min duration. During each 5-min activity session, activity counts (photobeam breaks) were recorded at 1-min intervals. Treatment groups were counterbalanced across test times.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

At necropsy, the adrenals, brain, liver, lungs, kidneys, ovaries, and testes (with epididymides) were weighed and recorded and organ/body and organ/brain weight ratios were calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment- related responses such as labored breathing and nasal discharge were seen during many of the exposures at all exposure levels. A slight increase in incidence was seen with increasing exposure concentration. Similar responses as well as moist rales were seen during the non-exposure periods during the 13 weeks of exposure. These responses abated during the 4-week recovery period.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the termination of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings which were considered to be related to the test material were seen in the nasoturbinal tissues (hypertrophy/hyperplasia of goblet cells in the respiratory mucosa and intra-cytoplasmic eosinophilic material in epithelial cells of the olfactory mucosa) of the 2 higher exposure levels and in the larynx (squamous/ squamoid metaplasia/ hyperplasia of the pseudostratified columnar epithelium covering the ventral seromucous gland) of all exposure levels. Minimal keratinization of the metaplastic epithelium of the larynx was seen in a small number of the 245 mg/m3 exposed rats. Some recovery was seen in both nasoturbinal and laryngeal tissues following the 4-week recovery period, but the recovery was incomplete.

Dose descriptor:

NOAEC

Effect level:

0.066 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: local effects: laryngeal keratinization

Dose descriptor:

NOAEC

Effect level:

0.245 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: no systemic effects at highest dose level

Critical effects observed:

not specified

Executive summary:

A study descrobed by TSCA, EPA September 1985 and May 1989 was performed.

The whole-body exposure of Sprague-Dawley rats to Caprolactam as an aerosol for 6 hours/day, 5 days/week for 13 weeks at analytically determined levels of 0.023, 0.066 and 0.245 mg/l resulted in transient clinical signs (secretory and respiratory) as well as respiratory tract effects (microscopically-detected nasoturbinal and/or laryngeal tissue changes) at all exposure levels with incomplete recovery within 4 weeks after exposure. However, these effects, except the laryngeal keratinization at 0.245 mg/l, were believed to be a normal physiological response to inhaled foreign particulate material and were not believed to represent an adverse response.This interpretation is fully supported by the proceedings of an international expert workshop of the European Society of Toxicology (Kaufmann et al., 2009, Exp. Tox. Path., 61, 591-603):slight laryngeal squamous metaplasia that are not observed diffusely could occur spontaneously or as treatment-induced lesions and should be assessed as “non-adverse”. Therefore, under the conditions of this study, a NOAEC (No Observed Adverse effect Level) was considered to be 0.066 mg/l due to effects on the upper respiratory tract. However, under the conditions of this study, a NOAEC was considered to be 0.245 mg/l due to no effects on the lower respiratory tract, systemic toxicity and neurotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

245 mg/m³

Study duration:

subchronic

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: described by TSCA, EPA September 1985 and May 1989

GLP compliance:

yes

Remarks:

(Huntington Life Science)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Caprolactam
- Physical state: white crystalline solid
- Analytical purity: 99.9%
- Stability under test conditions: stable for study duration
- Storage condition of test material: room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6 weeks
- Mean weight at study initiation: males 178 g (151-205 g), females 143 g (110-167 g)
- Housing: 2 per cage
- Diet (e.g. ad libitum): Certified Rodent Diet, No. 5002, PMI feeds Inc, MO.
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-27
- Humidity (%): 21-74
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

water

Remarks on MMAD:

MMAD / GSD: 2.9 µm/1.7

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: The placement of the animal in the whole body exposure chamber was rotated at each exposure to ensure uniform exposure of the animals.
- System of generating particulates/aerosols: A 1:1 solution, prepared by dissolving Caprolactam in an equal amount, by weight, of distilled demineralized water, was placed in a 250 ml Erlenmeyer flask and continuously stirred on a stirplate. The solution was fed directly, via an FMI Fluid Metering Pump, into the fluid inlet of a Spraying System, Inc. air atomizing nozzle (JCO-SS with a lA spray setup). The aerosol so generated was mixed with incoming house air by turbulence. Conditioned house air was metered into the control chamber.
- Temperature, humidity in air chamber: 21-32°C, 28-65%
- Method of particle size determination: Particle size distribution samples were withdrawn once during each exposure and determined using an aerodynamic particle sizer (TSI Aerodynamic Particle Sizer, Model 3300, and a Model 3302 TSI Aerosol Diluter). The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated.


TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, chamber concentration determinations were performed six times daily using gravimetric sampling. In addition, for each daily exposure group, one sample was also analyzed by high-pressure liquid chromatography (HPLC) using a Hewlett-Packard 1050 LC system and a UV detector.
- Samples taken from breathing zone: yes


VEHICLE (if applicable)
- Concentration of test material in vehicle: 1:1 aqueous solution with Caprolactam
- Lot/batch no. of vehicle (if required): Prepared by Pharmacy from water supplied by the Elizabethtown Water Company
- Purity of vehicle: Monthly analysis was provided by the Elizabethtown Water Company

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean (±SD) analytical exposure concentrations were 0±0, 23±4.9, 66±20 and 244±37 mg/m3 (approx. 0±0, 0.023±0.0049, 0.066±0.02 and 0.245 ±0.037 mg/l).
Gravimetrically determined exposure concentrations were (mean ±SD) 0.063 ±0.19, 24±5.6, 70±18 and 243±36 mg/m3 for the 13 weeks of exposure.

Duration of treatment / exposure:

13 weeks plus 4 week-recovery

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0 mg/L air (nominal)

Remarks:

0 mg/mL analytical conc.

Dose / conc.:

0.025 mg/L air (nominal)

Remarks:

0.023 mg/mL analytical concentration

Dose / conc.:

0.075 mg/L air (nominal)

Remarks:

0.066 mg/mL analytical concentration

Dose / conc.:

0.25 mg/L air (nominal)

Remarks:

0.245 mg/mL analytical concentration

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Post-exposure recovery period in satellite groups: An additional 10 animals/sex/group were similarly exposed and then held for a 4-week recovery period

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study and at least once during each exposure
- Cage side observations checked: mortality, general appearance and signs of toxic or pharmacological effects


BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly during study period and prior to the terminal and recovery sacrifices


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the first exposure and prior to the terminal sacrifice
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the exposure and recovery terminations
- Anaesthetic used for blood collection: Yes (light carbon dioxide anesthesia)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: erythrocyte count and morphology, total and differential leukocyte count, platelet count, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, prothrombin time, and activated partial thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the exposure and recovery terminations
- Animals fasted: Yes
- How many animals: all
- Parameters checked: serum alanine aminotransferase, serum aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, fasting glucose, total serum protein, albumin, globulin (calculated as total protein - albumin = globulin), albumin/globulin ratio (calculated), total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, and y-glutamyl transferase


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pretest and Weeks 5, 9, and 14 and Recovery (Week 19)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other: For homecage observations, each rat was observed and scored for posture, vocalization, and palpebral closure. On removal from the cage, each animal was observed and scored for ease of removal, ease of handling, lacrimation, appearance of fur, and salivation. Open-field evaluations included observations and scoring of gait, degree of locomotion, arousal, abnormal movements including tremors or convulsions, exophthalmia, piloerection, and presence or absence of diarrhea and urination. Reflex assessments consisted of scoring of responses to approach, tail pinch, finger snap, pupillary response, and air righting reflex. Additionally, landing foot splay and fore- and hindlimb grip strengths were assessed. Motor activity was monitored using an automated Photobeam Activity System (San Diego Instruments, Inc.). Sessions were 60 min in length; each session was divided into 12 intervals of 5-min duration. During each 5-min activity session, activity counts (photobeam breaks) were recorded at 1-min intervals. Treatment groups were counterbalanced across test times.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

At necropsy, the adrenals, brain, liver, lungs, kidneys, ovaries, and testes (with epididymides) were weighed and recorded and organ/body and organ/brain weight ratios were calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment- related responses such as labored breathing and nasal discharge were seen during many of the exposures at all exposure levels. A slight increase in incidence was seen with increasing exposure concentration. Similar responses as well as moist rales were seen during the non-exposure periods during the 13 weeks of exposure. These responses abated during the 4-week recovery period.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the termination of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings which were considered to be related to the test material were seen in the nasoturbinal tissues (hypertrophy/hyperplasia of goblet cells in the respiratory mucosa and intra-cytoplasmic eosinophilic material in epithelial cells of the olfactory mucosa) of the 2 higher exposure levels and in the larynx (squamous/ squamoid metaplasia/ hyperplasia of the pseudostratified columnar epithelium covering the ventral seromucous gland) of all exposure levels. Minimal keratinization of the metaplastic epithelium of the larynx was seen in a small number of the 245 mg/m3 exposed rats. Some recovery was seen in both nasoturbinal and laryngeal tissues following the 4-week recovery period, but the recovery was incomplete.

Dose descriptor:

NOAEC

Effect level:

0.066 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: local effects: laryngeal keratinization

Dose descriptor:

NOAEC

Effect level:

0.245 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: no systemic effects at highest dose level

Critical effects observed:

not specified

Executive summary:

A study descrobed by TSCA, EPA September 1985 and May 1989 was performed.

The whole-body exposure of Sprague-Dawley rats to Caprolactam as an aerosol for 6 hours/day, 5 days/week for 13 weeks at analytically determined levels of 0.023, 0.066 and 0.245 mg/l resulted in transient clinical signs (secretory and respiratory) as well as respiratory tract effects (microscopically-detected nasoturbinal and/or laryngeal tissue changes) at all exposure levels with incomplete recovery within 4 weeks after exposure. However, these effects, except the laryngeal keratinization at 0.245 mg/l, were believed to be a normal physiological response to inhaled foreign particulate material and were not believed to represent an adverse response.This interpretation is fully supported by the proceedings of an international expert workshop of the European Society of Toxicology (Kaufmann et al., 2009, Exp. Tox. Path., 61, 591-603):slight laryngeal squamous metaplasia that are not observed diffusely could occur spontaneously or as treatment-induced lesions and should be assessed as “non-adverse”. Therefore, under the conditions of this study, a NOAEC (No Observed Adverse effect Level) was considered to be 0.066 mg/l due to effects on the upper respiratory tract. However, under the conditions of this study, a NOAEC was considered to be 0.245 mg/l due to no effects on the lower respiratory tract, systemic toxicity and neurotoxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

66 mg/m³

Study duration:

subchronic

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Inhalation route:

In a 13-week inhalation study performed according to an EPA guideline (10/ rats/sex/dose), Sprague-Dawley rats were exposed to analytical concentration of 0, 0.023, 0.066 and 0.245 mg/l caprolactam (CAP) as an aerosol for 6 hours/day, 5 days/week (whole body exposure). The particle size was on average of 2.9 µm and consequently the test aerosol was highly respirable (Reinhold et al., 1997). No compound-related deaths occurred. There were no treatment-related responses observed in ophthalmic parameters, body weights, food consumption, neurobehavioral effects, organ weights, or macroscopic findings.

The only exposure related clinical observations were transient signs of respiratory tract irritation (e.g. nasal discharge, labored breathing). At the histological level the analysis of the respiratory tract revealed various changes compared to the control exposure group. Hypertrophy/hyperplasia of goblet cells in the nasal mucosa, intracytoplasmic eosinophilic material in the epithelium of the nasal mucosa and squamous/squamoid metaplasia/hyperplasia in the larynx mucosa were observed at all exposure levels. The severity of these changes increased in dose dependent manner and recovery was incomplete within the four weeks recovery period. These effects were interpreted to be local adaptive responses to the minimal irritant effect commonly associated with inhaled particulate material. This interpretation is fully supported by the proceedings of an international expert workshop of the European Society of Toxicology (Kaufmann et al., 2009, Exp. Tox. Path., 61, 591-603):slight laryngeal squamous metaplasia that are not observed diffusely could occur spontaneously or as treatment-induced lesions and should be assessed as “non-adverse”.

Keratinisation of the metaplastic epithelium in the larynx (reversible within 4 week recovery) was observed in the highest dose group. These changes were considered to be adverse indicating a NOAEC for local effects in the upper respiratory tract, of 0.066 mg/l.

Due to the aerodynamic diameter of the aerosol tested in this experiment (2.9 µm) it can be anticipated that a major part of the test substance is reaching the alveoli of the lung. 

No systemic toxicity was observed, indicating a NOAEC of 0.245 mg/l.

 

No further valid inhalation studies were identified.

 

Oral route:

 

A distinct gender variance in systemic toxicity of CAP was observed in a 90 day continuous feeding study with Sprague-Dawley rats (TNO, 1991). Ten rats per sex and dose were fed with dose levels of 0.05. 0.1, 0.25, 0.5 and 1 % (ca. 29, 60, 146, 296, 592 mg/kg bw in males, and 35, 70, 170, 342 and 704 mg/kg bw in females).

Decreased food intake and an increased relative liver weight were reported in the high dose group of female animals (NOEL females 342 mg/kg bw).

In males more severe sign of systemic toxicity were observed, indicated by an adverse increase of relative testes (18% at 1% CAP), liver (19% at 1% CAP) and kidney weights (25% at 1% CAP). Additionally a significant growth depression was described in the high dose group, which was paralleled by a decreased in food intake (decreased food efficacy).  

Kidney toxicity was confirmed by microscopically changes at 0.1 % and above, reflecting all typical signs of a hyaline-droplet degeneration. Based on the effects in the kidneys, the NOAEL for males was found to be 29 mg/kg bw/day.

A similar gender variance was observed in a second rat strain (Wistar rats). Again ten rats per sex and dose fed for 90-day with caprolactam in dose levels of 0.1, 0.3, 1.0 and 2.0 % (ca. 71, 203, 702, 1342 mg/kg bw in males and 77, 234, 785, 1459 mg/kg bw in females) (TNO, 1990). Decreased food intake in the high dose group and an increased relative liver weight in the two highest dose levels (14% at 785 mg/kg bw, 18% at 1459 mg/kg bw) were reported in female animals (NOAEL females 234 mg/kg bw). In males relative kidney, liver and thyroid weights were 18, 13 and 22 % increased in the 2% diet dose groups, respectively. Similar to SD-rats, nephrotoxicity was accompanied by histopathological indications of a hyaline dropled degeneration starting from the 0.3% dose level. Tubular nephrosis occurred to a higher degree and incidence in all treated male rats in comparison to the control. Due to the effects on the kidney, the NOAEL in males is considered to be slightly below ca. 71 mg/kg bw/d).

An oral feeding study was performed in order to analyze the sex and rat-strain specificity of the renal toxicity of caprolactam (Allied Corp., 1983). For this dose levels up to 613 mg/kg bw/d in Fischer-rats, 903 mg/kg bw/d in Sprague-Dawley and 968 mg/kg bw/d in Wistar rats were applied. Males were generally way more susceptible for this type of nephrotoxicity, with the first indications occurring at rather low dose levels. Furthermore it was shown, that the Fischer- and Sprague-Dawley strains are more susceptible than the Wistar-strain. An almost complete reversibility of the nephrotoxicity was observed in a recovery group reanalyzed 90 days after discontinued dosage.

 

These findings in kidneys are supposed to be of no relevance for other species including humans (Haschek and Rousseaux, Fundamentals of Toxicologic Pathology, Academic Press, 1996). Moreover, the subsequent 2 year cancer bioassay with rats and mice did not reveal any type of carcinogenicity or any impairment of kidneys (NTP, 1982).

 

Surprisingly, no comparable kidney toxicity was observed in a 3-generation fertility study with Fisher-rats (Serota et al., 1981). When the respective generations were fed for 10 week premating intervals with diets containing 100, 500 and 1000 mg/kg bw, significantly reduced body weight was noted in the high dose animals. Compound-related kidney effects only consisted of histomorphologic findings (slightly increased severity of spontaneous nephropathy, accompanied by the presence of granular casts) in three of the P1 males at the 1000 mg/kg bw level.

 

The effects of low levels of subchronic dietary Caprolactam on renal function were evaluated using both sexes and three rat strains (Fischer 344, Sprague-Dawley and Wistar) that received the test compound at diet levels of 0, 0.01, 0.05, 0.10 and 0.50% for at least 90 days (Allied Corp., 1983). Low doses of subchronic dietary CAP are capable of altering renal function when the system is stressed even at very low doses, i.e. 0.01%. The male animals were more susceptible to the effects of CAP than the females and the Fischer 344 and Sprague-Dawleys more so than Wistars. This effect of CAP appears to be more of a physiological alteration than a toxic or adverse effect, in that it is reversible and does not compromise the health of the animal.

 

In a NTP 13-week range-finding study (NTP, 1982) Fischer rats were fed with CAP dose levels of ca. 42, 83, 167, 333 and 500 mg/kg bw/d.

The only compound-related findings were decreased body weights in all treatment groups. In high dose group this decrease in body weight was accompanied by a significantly lower food consumption. Due to an absent dose relationship and a presumed palatability problem this finding can not be considered as adverse. No further compound-related effects were found, resulting in a NOAEL of 500 mg/kg bw/d based on the highest test concentration.

Similarly in B6C3F1 mice dose levels of ca. 813, 1667, 2500, 3333 and 5000 mg/kg bw/d in feed resulted in a body weight depression at all dose levels without a clear dose-response relationship. The NOAEL was identified as 5000 mg/kg bw/d since no further treatment related effects were reported.

In the associated chronic main studies dose levels of 187.5 and 375 mg/kg bw/d were assayed in rats and 1071 and 2143 mg/kg bw/d in mice. Consistently, weight gain depression, accompanied by decreased food intakes were the only treatment related observations. In these chronic studies this effect was considered as adverse since a dose dependency and a biological relevance was observed in the high dose group. Therefore the NOAELs were identified as 187.5 mg/kg bw/d for rats and 1071 mg/kg bw/d for mice.

Due to the restricted amount of parameters assayed in these chronic studies (e.g. clinical chemistry) in comparison to the OECD-guideline 408, these NTP studies can not be used in order to reliably identify an overall NOAEL for caprolactam.

 

Three groups of four male and four female beagle dogs each were fed diet containing Caprolactam at levels of 0, 0.1, 0.5 and 1 % (ca. 0, 32, 164, 292 mg/kg bw/d in males and ca. 0, 33, 158, 389 mg/kg bw/d in females) (Hazleton, 1980). A biologically irrelevant decrease in the mean body weights (2-7 % less) of the high-dose females was identified as the only treatment related effect. No treatment-related changes were noted with respect to clinical signs, food consumptions, clinical laboratory studies, ophthalmologic examination, organ weight data, or gross and microscopic pathology. Based on the results, the NOAEL for males and females was considered to be 292 and 389 mg/kg bw/d, respectively.

 

Combining all the data, CAP given by feed to rats only caused specific effects in the male rat kidney. This observation was shown to be related to a rat species which is supposed to have little or no relevance for other species, including humans. No systemic effects were identified in a subchronic dog sudy. Thus no classification as STOT is required.

 

Dermal route:

The only study available for the dermal route of exposure is of limited documentation (RL 4) and reduced scientific coverage (Hohensee, 1951). Four guinea-pigs each were dermally exposed (4 x 6 cm) to 5 and 10 % aqueous solution of Caprolactam daily for 62 days. No alterations were observed, though no quantitative assessment is possible since the applied volume of the test substance is not specified. 

 

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects in subchronic oral exposure and subchronic inhalation exposure experiments both performed in rats. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008,as amended for the fourteenth time in Regulation (EC) No. 2020/217.