2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Neurotoxicity

Description of key information

NOEL = 1000 mg/kg/day (male/female), rat, OECD 424, EPA OPPTS 870.6200, Johnson & Dryzga (2005)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2001 to 25 August 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The results from the neurotoxicity examinations are reported in limited detail, with the raw data and detailed conclusions reported in another study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

Deviations:

no

Principles of method if other than guideline:

Five animals per sex per dose were shared with the chronic repeated dose toxicity study performed as part of this study.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight on study day 1: 149.4 g for males and 113.4 g for females
- Housing: During acclimation animals were housed two-three per cage in stainless steel cages. During the main study, animals were housed two per cage in stainless steel cages. Cages had wire mesh floors and were suspended above catch pans. Cages contained a feed container and pressure activated nipple-type watering systems.
- Diet: ad libitum
- Water: ad libitum (municipal water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 - 24.7 °C
- Humidity: 40-69 % (relative)
- Air changes: 12-15 changes per hour
- Photoperiod: 12 hour light/dark photocycle

IN-LIFE DATES
- From: Test material administration began on the 14 August 2001 for the males and the 15 August 2001 for the females
- To: Necropsies took place on the 15 to the 21 August 2002

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diet mixes were fed to the animals for a 2-week period before a new diet mix was prepared.
- Mixing appropriate amounts with (Type of food): 7 % premix was mixed with the diet to form the test diets.
- Storage temperature of food: Ambient temperature in sealed vessels.

Diets were prepared by serially diluting a concentrated test material- feed mixture (premix) with ground feed. Premixes were mixed periodically throughout the study based on stability data. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. Subsequently, the concentrations of the test material in the feed were adjusted weekly for the first 13 weeks of the study and at four-week intervals thereafter, based upon the most recent body weight and feed consumption data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the premixes and all the mixes, including the control diet, were performed prior to dosing and at approximately 4, 8 and 12 months of the study. The test material was extracted with solvent and analysed using liquid chromatography-mass spectrometry (LC-MS).
The homogeneity of the mixtures was determined in the low dose diet for the females and the high dose male test diet. The analyses were performed prior to dosing and then again at around 4, 8 and 12 months. Additional homogeneity determinations were performed when the pre-mix concentration was changed from 3 to 7 %.
Data on stability was available from a previous four-week study performed in mice which demonstrated that the test material was stable for at least 21 days in rodent chow. An additional stability test for the 7 % premix study was conducted up to 55 days.
One reference sample per sex per dose per mix were retained and stored at ambient temperature in sealed vials.

RESULTS
The concentrations of the test material were determined for the control and test diets from nine time points and were found to be acceptable. The mean concentrations for each dose level over the course of the study ranged from 96.9 to 105 % of targeted concentration.
No test material was found in the control diet. LC-MS analysis of each individual sample indicated 78.4-121 % of the target concentration, with the exception of one value of 135 %. A follow-up analysis using the same diet mixing instructions resulted in 90.7 % of the target concentration.
The homogeneity in rodent feed was determined for nine separate mixing batches for the 5 mg/kg/day female and 1000 mg/kg/day male test diets. In addition, homogeneity was conducted on the 50 and 1000 mg/kg/day female diet mixes and the 7 % premix and 1000 mg/kg/day female diet mix. The homogeneity of the diets was considered acceptable, with relative standard deviations for all diets sampled between 2.33 and 15.9 %, with the exception of one analysis where the relative standard deviation was 43.71 % for a sample from the 5 mg/kg/day female dose group. The relative standard deviation for the majority of samples was < 10 %.
Stability of the test material was determined in the 7 % premix and was determined to be stable in the premix for at least 55 days, at which it was 96.5 % of the concentration found initially.

Duration of treatment / exposure:

12 months

Frequency of treatment:

Daily; continuous in the diet

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The high dose was selected based on preliminary results of a subchronic dietary toxicity study with a four week recovery period. The cecal weights were around 2.8 times the control values for males and females dosed with 1000 mg/kg/day for 13 weeks. Males had very slight epithelial hyperplasia of the cecum and ileum. The high dose selected was therefore expected to produce increased weight of the cecum and a decreased urine pH. The other doses were expected to provide a dose response relationship for any effect that might be observed at the highest dose tested for this study. The low dose was expected to produce a NOEL.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for mortality and moribundity at least twice daily. Other observations were carried out at least once a day.
- Cage side observations: Cage side observations included but were not limited to decreased/increased activity, repetitive behaviour, vocalisation, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency and faecal/urinary quantity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-exposure then monthly until termination

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-exposure, weekly for the first 13 weeks and then monthly thereafter.

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

FOOD EFFICIENCY:
- Calculated as (g/feed consumed/day)/( g bodyweight gain): Yes (first 13 weeks of the study)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure then at termination using indirect ophthalmoscopy
- Dose groups that were examined: All groups

OTHER: Haematology, clinical chemistry and urinalysis investigations were also carried out on the subset of animals shared with the chronic toxicity group.

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined: Rectal temperature, grip performance, landing foot splay, stimulus response and physical examination.
- Minimisation of bias: Performed under red light at approximately the same time each day
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Site of testing: Cage side, hand held and open field observations were performed.
- Time schedule for examinations: 1, 3, 6, 9 and 12 months.
- Environmental conditions: Performed under red light
- Description of equipment where required: Open field observations were performed in a clear plastic box (50 x 50 cm). Rectal temperature was measured using a rectal thermistor (Physitemp) placed approximately 4 cm into the rectum for 15-20 seconds. Grip performance was evaluated using an electronic strain gauge. For the examination of hindleg grip strength, the animals are placed with the forelegs on a plastic bench, and the hind feet are placed on a plastic screen. For the examination of forelimb grip strength, the animals forefeet were placed on a plastic screen, with their hindlegs suspended approximately 10 cm above a plastic platform. To measure landing foot splay, the outermost toes of the animals on both hindfeet are inked and the animals are dropped from a height of 30 cm onto a recording sheet. The animals are re-inked between trials and the process is repeated twice.

LOCOMOTOR ACTIVITY: Yes
- Replicates used: 6
- Type of equipment used: Motor activity cages (infra-red beams)
- Length of session, number and length of subsessions: 8 minute long sessions resulting in 48 minutes total per test session
- Parameters measured: Whenever a rat crossed in front of a beam, an interruption of the beam was recorded. If the beam was broken for more than 100 msec following an inter-beam break duration that is greater than 100 msec, then the break is recorded as an activity count. Motor activity was recorded as the square root of activity counts (to minimise problems of heterogeneity of variance and departure from normality).
- Ambulatory activity: The minimum beam break parameters were designed to remove any interference from movements such as head bobbing and tail flicking.

Sacrifice and (histo)pathology:

- Time point of sacrifice: 12 months
- Number of animals sacrificed: 5 (high-dose and control group animals)

PARAMETERS MEASURED
- Brain weight: Yes
- Procedures for perfusion: Rats were given an intraperitoneal injection of 0.2 mL of heparin (10 000 USP/mL) per 100 g bodyweight 10 minutes prior to perfusion. Rats were deeply anaesthetised and the heart exposed. The left ventricle was cannulated and the right atrium was incised. Rats were perfused by gravity pressure with 0.05 M phosphate buffer containing sodium nitrite followed by a phosphate buffered solution containing 1.5 % glutaraldehyde – 4 % formaldehyde (c. 540 mOs).
- Number of animals perfused: 5 from the high dose group and 5 from the control group
- Tissues evaluated: Brain (evaluated in nine sections), head, spinal column with spinal cord, for- and hindlimbs and tail were trimmed to remove excessive skin and muscle. Muscle from the hindlimbs was reflected further to expose the nerves. The thoracic and abdominal viscera were also preserved.
- Type of staining: Haematoxylin and eosin was used to stain the brain (olfactory bulb, cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, pons, cerebellum, and medulla oblongata), trigeminal ganglion and nerve (bilateral), pituitary gland, eyes with optic nerves (bilateral), spinal cord (cervical and lumbar), olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial).
Toluidine blue was used to stain the spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial [proximal and distal - at the knee and calf muscle branches] and sural),
- Methodology of preparation of sections:
The brain (olfactory bulb, cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, pons, cerebellum, and medulla oblongata), trigeminal ganglion and nerve (bilateral), pituitary gland, eyes with optic nerves (bilateral), spinal cord (cervical and lumbar), olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial) were all prepared using standard histologic procedures.
Spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial [proximal and distal - at the knee and calf muscle branches] and sural) were osmicated prior to embedding.
- Thickness:
6 µm - brain (prepared as nine sections including the olfactory bulb, cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, pons, cerebellum, and medulla oblongata), trigeminal ganglion and nerve (bilateral), pituitary gland, eyes with optic nerves (bilateral), spinal cord (cervical and lumbar), olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial).
2-3 µm - Spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial [proximal and distal - at the knee and calf muscle branches] and sural).
- Embedding media: 6 µm in paraffin, 2-3 µm in epoxy resin
- Number of animals evaluated from each sex and treatment group: 5 animals per sex in the high dose and control groups. The other groups were necropsied if needed to establish a NOEL.

Statistics:

Bodyweights (FOB timepoints), grip performance, rectal temperature, landing foot splay, motor activity and FOB observations. The FOB observations were converted to ranked scores (males and females at each dose) and converted into average scores.
The incidence FOB observations were analysed by a z-test of proportions comparing each treatment to the control. Means and standard deviations were calculated for all continuous data and homogeneity of variance was evaluated with the Bartlett’s test (α= 0.01). If significant, the data was transformed to obtain equality of the variances. The data was reviewed and an appropriate form was selected. This was then subjected to an appropriate parametric analysis.
Bodyweights, rectal temperature, grip, landing foot splay, and motor activity were analysed by a factorial repeated-measure design, multivariate approach, with factors of sex and treatment and the repeated factor of time. The statistical significance of the treatment-by-time-by-sex interaction was first tested by repeated measure ANOVA. If significant, the analysis was repeated by sex (α = 0.05). Treatment-by-time and treatment-by-time-by-epoch were then examined. If significant, linear contrasts were calculated to determine which dose differed from the control, Pillai Trace statistic was used to evaluate significance (α = 0.02). Further analyses (α= 0.01) were performed at the study director’s discretion.
Due to numerous statistical comparisons in the same group, false positive rates were expected to exceed alpha. Therefore, the toxicologic interpretation considered dose-response, biological plausibility and consistency, and historical controls. Feed consumption was evaluated by Bartlett's test for equality of variances. Based on the outcome, exploratory data analysis was performed by a parametric or nonparametric analysis of variance (ANOVA). If significant (α = 0.05), the ANOVA was followed by Dunnett's test or Wilcoxon Rank-Sum test with a Bonferroni correction.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no statistically significant or treatment related changes in clinical or detailed clinical observations in any treated group. Periocular soiling was found in one-two animals per sex per dose, however this was found to be sporadic and not attributed to treatment with the test material.

Mortality:

no mortality observed

Description (incidence):

No statistically significant differences in mortality rates for males and females at any dose group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower for males and females at 1000 mg/kg and lower for males at 500 mg/kg. see details on effects below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Although feed consumption was highly variable, no clear pattern emerged in relation to administration of the test material.
Feed efficiency decreased over the 13-week time period in both sexes and all dose groups due to slowing of the growth rate while maintaining relatively constant feed consumption. Although slightly increased feed consumption was noted for males given 1000 mg/kg/day and a non-dose related increase for all female dose groups, these minor changes did not result in a consistent pattern of treatment-related altered feed efficiency.

Food efficiency:

no effects observed

Description (incidence and severity):

Although feed consumption was highly variable, no clear pattern emerged in relation to administration of the test material.
Feed efficiency decreased over the 13-week time period in both sexes and all dose groups due to slowing of the growth rate while maintaining relatively constant feed consumption. Although slightly increased feed consumption was noted for males given 1000 mg/kg/day and a non-dose related increase for all female dose groups, these minor changes did not result in a consistent pattern of treatment-related altered feed efficiency.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ocular haemorrhage, engorged blood vessels, pale fundus, cloudy cornea, periocular soiling, cloudy lens, opaque cornea, opaque lens, phthisis bulbi, missing eye and enlarged or protruding eye were observed. These were not treatment related due to their low incidence and lack of dose-response. Periocular soiling was considered to be a non-specific clinical sign, while eyes with pale fundus, opaque cornea, opaque lens, cloudy cornea or cloudy lens were considered to be age related changes. Phthisis bulbi and missing eye were considered to be secondary to disease or blood collection.

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The cecum of all males and females dosed 1000 mg/kg/day and 9 males and 6 females dose at 500 mg/kg/day was found to be grossly enlarged at study termination. With the exception of optic nerve lesions which were attributed to repetitive retro-orbital bleeding for blood samples, all other observations (including observed masses) were found to be sporadic and not test material related.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No effects were noted in the FOB, motor activity test and neurohistopathology examinations.

Details on results:

BODY WEIGHT AND WEIGHT GAIN
Bodyweights for males dosed with 500 and 1000 mg/kg/day were lower than controls. The difference between treated animals and control developed gradually during the study and reached statistical significance at day 120 where the males at 500 and 1000 mg/kg/day weighed 2.4 and 3.8 % less than controls, respectively. These groups were generally statistically identified for the remainder of the study. Males at 50 mg/kg/day had slightly lower bodyweights than controls, however this was not attributed to treatment as they were within 97 % of the control bodyweights and were only transiently statistically identified. Males at 5 mg/kg/day had slightly lower bodyweights, but this was not statistically identified at any point. The initial bodyweights of male rats dosed with 5 or 50 mg/kg/day were slightly lower than the rats in the other dosing groups.
Bodyweights for females dosed with 1000 mg/kg/day were slightly lower than the controls and remained so throughout the study. These were considered to be treatment related. The decrement developed gradually and was maintained at approximately the same level throughout the study. This was statistically identified at day 36 (2.0 % lower than controls) and remained 2-3 % lower throughout the study, but were identified as statistically significant at around half the time.
These differences in males and females were concurrent with lower bodyweight gains at 1000 mg/kg/day for males and females and males at 500 mg/kg/day. At 12 months, gains were 4.5 and 7.7 % lower for 500 mg/kg/day and 1000 mg/kg/day males, respectively. Bodyweights for females dosed with 1000 mg/kg/day were 2.8 % lower than controls at termination.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed in any neurotoxicological examination. The NOEL is therefore considered to be 1000 mg/kg/day, the highest dose tested.

Conclusions:

Under the conditions of the test, no chronic neurotoxic effects were observed in male and female rats. The NOEL for neurotoxicity was therefore considered to be 1000 mg/kg/day, the highest dose tested.

Executive summary:

The chronic neurotoxicity of the test material was evaluated in a study conducted in accordance with the standardised guidelines OECD 424 and EPA OPPTS 870.6200 under GLP conditions as part of a combined chronic repeated dose toxicity study and carcinogenicity study.

Male and female Fischer 344 rats (10 per sex per dose, five of which from each dose were shared with the chronic repeated dose toxicity study) were exposed to the test material in the diet at doses of 0 (control), 5, 50, 500 and 1000 mg/kg/day for 12 months.

The animals were subjected to functional observation battery (FOB) and motor activity examinations at 1, 3, 6, 9 and 12 months. At the end of the study, the five animals which were not shared with the chronic repeated dose toxicity study were necropsied and subjected to a detailed neurohistopathological exam.

Under the conditions of the test, no chronic neurotoxic effects were observed in male and female rats. The NOEL for neurotoxicity was therefore considered to be 1000 mg/kg/day, the highest dose tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

Due to the limited reporting of the results of the neurotoxicity investigation in the study available, the quality of the database is considered to be average.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the key study Johnson & Dryzga (2005), the chronic neurotoxicity of the test material was evaluated in a study conducted in accordance with the standardised guidelines OECD 424 and EPA OPPTS 870.6200 under GLP conditions as part of a combined chronic repeated dose toxicity study and carcinogenicity study.

Male and female Fischer 344 rats (10 per sex per dose, five of which from each dose were shared with the chronic repeated dose toxicity study) were exposed to the test material in the diet at doses of 0 (control), 5, 50, 500 and 1000 mg/kg/day for 12 months.

The animals were subjected to functional observation battery (FOB) and motor activity examinations at 1, 3, 6, 9 and 12 months. At the end of the study, the five animals which were not shared with the chronic repeated dose toxicity study were necropsied and subjected to a detailed neurohistopathological exam.

Under the conditions of the test, no chronic neurotoxic effects were observed in male and female rats. The NOEL for neurotoxicity was therefore considered to be 1000 mg/kg/day, the highest dose tested.

Justification for selection of effect on neurotoxicity via oral route endpoint:
A single good quality study was available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to repeated dose toxicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to repeated dose toxicity.