Titanium(4+) ethanolate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Well reported study following NTP Protocol.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

P generation no organ weights, sperm parameters or oestrous cycle included; P animals slightly older than recommended at first exposure; low number of pregnant females

Qualifier:

according to guideline

Guideline:

other: NTP Protocol. Fertility assessment by continuous breeding

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratories
- Age at study initiation: (P) animals 6 weeks at receipt, 11 weeks at first exposure.
- Fasting period before study: no
- Housing: 4-5 per cage by sex. In pairs during breeding and thereafter individually for 21 days.
- Diet (e.g. ad libitum): Pelleted feed (NIH-07 open formula rodent chow) ad libitum
- Water (e.g. ad libitum): deionized/filtered ad libitum
- Acclimation period: 2 weeks prior to preliminary range-finding study


ENVIRONMENTAL CONDITIONS
- Temperature : approx 21 C
- Humidity (%):
- Air changes (per hr): 12-14
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Ethanol administered in deionized, filtered water.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: litters were proof of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of ethanol formulation in drinking water, control drinking water and bulk chemical were sent to Midwest Research Institute (Kansas City, MO), prior to preliminary range finding study, and at weeks 1, 6, 12, and 18 of main study with Parental animals.

Duration of treatment / exposure:

Exposure period: 18 weeks
Premating exposure period (males): Parental 7 days; F1 74 days
Premating exposure period (females): Parental 7 days; F1 74 days

Frequency of treatment:

ad libitum

Details on study schedule:

Number of generation studies: 2

Remarks:

Doses / Concentrations:
5, 10 and 15% v/v in water
Basis:
nominal in water

No. of animals per sex per dose:

20 for P generation, also 20 F1 animals at the high dose mated at 74 days old.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 14 day dose range finding study conducted. High dose for the 13 week study chosen such that depression of weight gain <10%

Positive control:

N/A

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: No. Only twice daily cage side inspections.

BODY WEIGHT: Yes
- Time schedule for examinations: at end of week 1, 2, 5, 9, 13 and 18.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: at end of week 1, 2, 5, 9, 13 and 18.

Oestrous cyclicity (parental animals):

Gestation index, changes in lactation and changes in oestrus cycles were not studied.

Sperm parameters (parental animals):

Epididymal and vas sperm were evaluated for concentration, motility and morphology in F1 males only.

Litter observations:

Litters were not standardized.

Postmortem examinations (parental animals):

Not conducted

Postmortem examinations (offspring):

GROSS NECROPSY
- High dose F1 animals had liver, kidney/adrenal and male sex organs weighed at termination.

Statistics:

Fertility and mating indices: Cochran-Armitage test for dose related trend and Fisher's exact test for comparisons between groups.
Size and number of litters, proportion of live pups and sex ratio, pup body weight, necropsy weight and sperm characteristics: Kruskal-Wallis for overall differences among groups, Jonckheere's test for dose related trends and Wilcoxon's test for pairwise tests.
Litter and dam weight: Williams' test

Reproductive indices:

Fertility indices were 97, 100, 100 and 94% in the controls and 5%, 10%, 15% ethanol groups respectively.
The F1 offspring of the 15% ethanol pairs had fewer live pups per litter. Unadjusted F1 live pup weight was greater for females and combined sexes at 5% but not at the higher concentrations. Body weights were lower than control in the 15% ethanol treated F1 offspring at mating and on day 21. Fertility indices in F1 matings were 85% and 65% in the controls and 15% ethanol groups respectively. Their F2 offspring weighed less as ethanol treated pups than control pups (males, females or both sexes). Other reproductive performance indices e.g. gestation index, changes in lactation and changes in oestrous cycles were not studied.

Offspring viability indices:

Proportion of pups born alive

Clinical signs:

not examined

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality in P animals is reported but not discussed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights at week 13, 38.4+/-0.6 (5, 10 and 15% ethanol) and 39.6+/-0.6g (control); for females 3.4% lower in at 15% ethanol compared with control at week 13.

No food consumption determined only water consumption: Daily water consumption at week 13, 7.0+/-0.1 g per mouse for controls, 7.1+/-0.2g for 5% group, 6.4+/-0.2g for 10% and 5.3+/-0.2g for 15%.

Dose descriptor:

NOAEL

Effect level:

15 other: % in drinking water

Sex:

male/female

Basis for effect level:

other: No effects observed in parameters studied at all doses

Clinical signs:

not examined

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- Not reported. Litters born to P at 15% ethanol had reduced number of live pups per litter.

BODY WEIGHT (OFFSPRING)
- Litter size and weights were not given. Pups born in final F1 generation of animals exposed to 15% ethanol pre- and post-natally weighed less than controls at birth and days 21 and 74.

SEXUAL MATURATION (OFFSPRING)
- Sex ratios: Not influenced by treatment

ORGAN WEIGHTS (OFFSPRING)
F1 males from the 15% group at adulthood had decreased bodyweight and and decreased weight of testis and epididymides and seminal vesicles. In F2 females, relative liver and kidney/adrenal weights were increased.
- Vaginal opening or preputial separation: Not studied.
- Anogenital distance: Not measured.

GROSS PATHOLOGY (OFFSPRING)
- Not examined.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 other: % in drinking water

Sex:

male/female

Basis for effect level:

other: At the highest dose fewer pups per litter were observed and significant changes to sperm motility

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

< 15 other: % in drinking water

Sex:

male/female

Basis for effect level:

other: Lower live pup weight observed at the 15% dose studied

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Reproductive effects observed:

not specified

Result: No observed effect on fertility.

Parental/F1 data: Ethanol treatment had no effect on bodyweights and on the proportion of breeding pairs producing at least 1 litter during the continuous breeding phase or the number of litters per pair. 

Effects on sperm and male reproductive organs: In the F1, 15% ethanol group there was a significantly decreased % motile sperm but no changes in sperm concentration, % abnormal sperm or % tailless sperm. There was a significant decrease in testis, epididymis and seminal vesicle weight but not when adjusted for body weight.

Post natal survival until weaning: Not reported.

Estimated daily intakes were 0, 6.9, 13.8 and 20.7g/kg ethanol.

Conclusions:

Overall, ethanol in drinking water at concentrations up to 15% (equivalent to 20.7 g/kg/day) had no demonstrable effect on fertility in this two-generation study.

Executive summary:

A two-generation study investigated the effects of 5%, 10% and 15% ethanol in drinking water in reproduction and fertility. Male and female CD-1 mice were continuously treated for 1 week prior to mating and for a 14 week breeding period followed by a 21 day holding period when they were separated and housed individually. The F1 offspring of the 15% ethanol pairs had fewer live pups per litter but ethanol treatment had no effect on the proportion of breeding pairs producing at least 1 litter during the continuous breeding phase or the number of litters per pair. The F1 offspring from the 15% group had decreased bodyweight at weaning and mating, and a decreased weight of testis, epididymides and seminal vesicles which was no longer evident when these were adjusted for body weight. There was also a significantly decreased percentage motile sperm but no changes in sperm concentration, and percentage of abnormal sperm or tailless sperm. When reproductive performance of F1 control and 15% ethanol-treated breeding pairs was assessed at 74 days of age, there was no significant difference in mating and fertility between the groups. However, adjusted live pup weight for the ethanol group was significantly reduced compared to controls which was likely due to generalized maternal toxicity.

Reference 2

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to conclude results reliable for use in hazard assessment. Limited experimental detail provided. Study incomplete as a comprehensive assessment of effects on male fertility.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

short paternal exposure period; no pathology conducted; number of pregnant females low; male treated rats mated to untreated females.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 400-500g males; 200-300g females
- Housing: Females housed individually.

ENVIRONMENTAL CONDITIONS
- Temperature: 73 +/- 3F.
- Humidity: 40-50%.
- Light/dark cycle (hrs): 12/12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air in chamber

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure was conducted in 0.5m3 chambers with dynamic air flow
- Air change rate: (one air change per minute.)

Details on mating procedure:

After 2 day non-exposure period males were mated individually with untreated virgin females. Mating period 5 days. Mating confirmed by presence of sperm plugs under cages or vaginal smears.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 7 hours /day
Premating exposure period (males): 6 weeks
Premating exposure period (females): none
Postmating exposure period (females): Days 1-20 gestation
Duration of test: see method details

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 10000, 16000ppm
Basis:
nominal conc.

No. of animals per sex per dose:

18/group male
15/group female (pregnant)

Control animals:

yes

Details on study design:

Analytical monitoring: Yes (IR analyser) - exposure found to be within 11% of nominal). Independently cross-checked with charcoal adsorption tubes.

Parental animals: Observations and examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food intake were measured.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes

Litter observations:

Pups were individually weighed weekly for five weeks.

Dose descriptor:

NOAEL

Effect level:

> 16 000 ppm

Sex:

male

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Generation:

F1

Sex:

not specified

Remarks on result:

not determinable because of methodological limitations

Reproductive effects observed:

not specified

Result: negative

No effect on weight gain, feed or water intake.  No effect on fertility or litter sizes.  Offspring numbers averaged 14/litter. Previous studies quoted as showing exposures to 10000 and 16000 ppm (30 400mg/m3) ethanol typically give rise to blood ethanol concentrations of 30 and 500 mg/l ethanol.  Authors calculate that for rats exposures in excess of 11000 ppm are required to begin accumulating ethanol in the blood and that ethanol is no more toxic by the inhalation route than by other routes.

No data on offspring body weight gain provided but it is presumed that there was no change from controls.

6 weeks treatment period for males does not cover one complete cycle of spermatogenesis.

Conclusions:

Ethanol treatment did not affect the fertility of male rats determined by number of litters and litter size.

Executive summary:

Male rats were exposed to ethanol in an inhalation chamber at concentrations of 11,000ppm or 16,000ppm for six weeks. After a two day non-exposure period they were mated to untreated females for 5 days. The pregnant females received the same experimental exposure from day 1 -20 of gestation and were then allowed to deliver their litters. Ethanol treatment did not affect fertility or litter sizes.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

13 800 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 400 mg/m³

Study duration:

subacute

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Testing is not necessary since there is enough evidence on the effects of the main decomposition products on fertility to conclude that titanium(4 +) ethanolate is not toxic to reproduction. As the substance hydrolyses rapidly (<5min) when it comes in contact with water or moisture (Brekelmans, M.J.C., 2013), intrinsic properties are related to the degradation products. After hydrolysis no significant reaction products other than ethanol and non-hazardous hydrated titanium dioxide exist.

Ethanol has harmonized classification according to EU regulation No. 1272/2008 (CLP) and it is not classified as toxic to reproduction. There are several studies available for ethanol to evaluate the reproduction toxicity of the substance. The other decomposition product of titanium(4+) ethanolate is non-hazardous TiO2. Thus, it is concluded that there is no relevance to further evaluate TiO2 in the chemical safety assessment.

In the following sections studies related to reproductive toxicity of ethanol are discussed in more detailed.

A two-generation study investigated the effects of 5% (corresponding to a dose of about 6.9 g ethanol/kg bw/day), 10% (13.8 g ethanol/kg bw/day) and 15% (20.7 g ethanol/kg bw/day) ethanol in drinking water in reproduction and fertility (George et al., 1985). Male and female CD-1 mice were continuously treated for 1 week prior to mating and for a 14 week breeding period followed by a 21 day holding period when they were separated and housed individually. The NOAEL for maternal and paternal toxicity was established to be 15% ethanol in drinking water (20.7g/kg bw/day). The F1 offspring of the 15% ethanol pairs had fewer live pups per litter but ethanol treatment had no effect on the proportion of breeding pairs producing at least 1 litter during the continuous breeding phase or the number of litters per pair. The F1 offspring from the 15% group had decreased bodyweight at weaning and mating, and a decreased weight of testis, epididymides and seminal vesicles which was no longer evident when these were adjusted for body weight. There was also a significantly decreased percentage motile sperm but no changes in sperm concentration, and percentage of abnormal sperm or tailless sperm. When reproductive performance of F1 control and 15% ethanol-treated breeding pairs was assessed at 74 days of age, there was no significant difference in mating and fertility between the groups. However, adjusted live pup weight for the ethanol group was significantly reduced compared to controls which were likely due to generalized maternal toxicity. The NOAEL for F1 toxicity was 10% ethanol (13.8g/kg bw/day) in drinking water based on the observation on fewer pups per litter and changes in sperm motility.

In the other study, male rats were exposed to ethanol in an inhalation chamber at concentrations of 11 000 ppm or 16 000 ppm for six weeks (Nelson, et al, 1985). After a two day non-exposure period they were mated to untreated females for 5 days. The pregnant females received the same experimental exposure from day 1 -20 of gestation and were then allowed to deliver their litters. Ethanol treatment did not affect fertility or litter sizes and the weight gain of parental animals. Incidence of fertility did not differ from controls and no group differences were found for litter size, number of dead pups, or length of pregnancy. Offspring survival and weight gain was also not affected by ethanol treatment. As conclusion, the NOAEL for paternal toxicity was > 16 000 ppm since no effects on fertility was noted at highest dose studied.  

Short description of key information:
The weight of evidence on decomposition of titanium(4+) ethanolate and studies from hazardous hydrolysis product, ethanol, indicates that this substance has no effects on fertility.

Justification for selection of Effect on fertility via oral route:
No studies available for the target substance which is highly reactive. Data is obtained from the most reliable two-generation study available for the main decomposition product (ethanol).

Justification for selection of Effect on fertility via inhalation route:
No studies available for the target substance which is highly reactive. Data is obtained from the reliable study performed for the main decomposition product (ethanol).

Justification for selection of Effect on fertility via dermal route:
Not likely route of exposure.

Effects on developmental toxicity

Description of key information

The weight of evidence on decomposition of titanium(4+) ethanolate and studies from the hazardous hydrolysis product, ethanol, indicates that this substance has no effects on development.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Not a guideline study and publication does not report whether study performed according to GLP. However, data appears to be well documented and scientifically acceptable. Read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture complete hydrolysis will take place with no significant reaction products other than alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the toxicokinetics of target substance. Because of the rapid hydrolysis, the influence of the mode of administration through inhalation, dermal and oral is related to the hazardous degradation product (alcohol) released from the target substance. The identification of degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance, which might change the hazardous properties of the target substance compared to the properties of the pure alcohol. As there is a mechanistic reasoning to the read-across, the unnecessary animal testing is avoided by using the read-across data from the degradation product (relevant alcohol) to evaluate irritation, sensitization and the short term and long-term toxicological effects and mutagenicity of the target substance.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ethanol was added to the diets of female rats for six weeks (from 3 weeks prior to mating to gestational day (GD) 21) in an attempt to determine effects on fetal growth and skeletal development.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, St. Constant, Quebec
- Age at study initiation: 3 months
- Weight at study initiation: mean 264 g (238-283 g)

ENVIRONMENTAL CONDITIONS
- Temperature-controlled rooms
- Photoperiod (hrs dark / hrs light): 12/12 (artificial lighting between 06.00-18.00)

Route of administration:

other: liquid diet

Vehicle:

unchanged (no vehicle)

Details on exposure:

no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Details on mating procedure:

Following 3 weeks of ethanol exposure, the virgin female rats were placed in breeding cages with unexposed males. Average time for mating was 3 days (range 1 - 5 days), and rats recieved lab chow ad libitum during this period. The appearance of a vaginal plug was considered day 1 of gestation (GD 1).

Duration of treatment / exposure:

Females exposed for six weeks (3 weeks prior to mating until GD 21)

Frequency of treatment:

Daily (liquid diet presented at 17.00)

Duration of test:

6 weeks

No. of animals per sex per dose:

Three test groups, fed diets containing 15% (E15; n=15), 25% (E25; n=15), and 36% (E36; n=26) ethanol-derived calories.
Four control groups, three of which were pair-fed (PF) isocaloric controls with maltose-dextrose replacing the ethanol at 15% (PF15; n=15), 25% (PF25; n=15), and 36% (PF36; n=25), and an additonal control group (C; n=31) receiving liquid control diet ad libitum.

Control animals:

other: Pair-fed (PF) isocaloric liquid diet (with the ethanol calories replaced by maltose-dextrose)... (see attached file)

Details on study design:

- Dose selection rationale: Dams were fed ethanol at doses to approximate low, moderate and high levels of maternal drinking.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout experiment

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: Number of fetuses per litter counted

Fetal examinations:

- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [all per litter] The percent ossification of the fetal bones (scapula, humerous, ulna, radius, femur and tibia) were measured using a Zeiss dissecting microscope with a linear eyepiece raticule. The number of ossification centres was determined for the sternum, sacrum, and metatarsals.
- Head examinations: No data

Statistics:

Differences between the groups regarding the effects of diet and dose of ethanol were determined by one- and two-way ANOVAs followed by Newman-Keuls post hoc tests, except for the number of ossification centres which was determined by one- and two-way Kruskal-Wallis tests followed by a Tukey-like nonparametric mulitple comparison test. Comparisons among diets were analysed by seperate one-way Kruskal-Wallis tests for each of the exposure levels. Repeated measures ANOVAs were used to determine the effects of week of gestation on maternal ethanol intake (wk 0, 1, 2 and 3) and on peak blood ethanol concentrations (BEC). An unpaired t test was used to compare terminal BEC samples between E25 and E36 dams.

Indices:

Ponderal index (fetal weight/length3) calculated to examine effect of ethanol on relationship between fetal body weight and length.

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Overall, there was no statistically significant difference in maternal weight gain between the ethanol (E15, E25 and E36) and corresponding pair-fed (PF15, PF25 and PF36) dams. However, there was a significant decrease in weight gain at the E36, compared to E15 and E25 dams.

Dose descriptor:

LOAEL

Effect level:

8 200 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

5 200 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No significant differences in the number of fetuses per litter between the groups were apparently observed.

Maternal ethanol intake from the E36 mothers resulted in fetuses which were statistically significantly lighter than the pair-fed (PF36) isocaloric controls, the ad libitum controls, and compared to the E15 and E25 groups. In addition, fetuses from the E36 dose groups were significantly shorter than the ad libitum controls and the E25 and E15 groups. The ponderal index was not significantly affected by diet or dose, indicating that the ethanol-induced restriction in growth did not alter the relationship between body weight and length. No significant effects on fetal growth (body weight or length) were seen in the fetuses of mothers exposed to E15 or E25 compared to the PF15 or PF25 isocaloric controls, respectively, or compared to the ab libitum controls.

In the E25 group, a statistically significant effect on fetal bone ossification was seen for the ulna, radius, tibia, and scapula, but not the sternum, metatarsals, sacrum, femur or humerus, when compared to the PF25 isocaloric controls. No significant differences between ossification at the E15 compared to the PF15 isocaloric controls were seen for any of the bones sites, although a statistically significant effect at the E15 was found for the radius compared with the ab libitum controls only.

Ethanol induced a statistically significant delay in the development of body weight and ossification of individual bones, particularly at the E36 dose group. The radius and scapula showed the greatest delay at the E25 and E36 groups, followed by the ulna and tibia.

Taken together, the data suggest that prenatal ethanol exposure (at 8.2 g/kg bw/day and above), caused a delay in the early development (ossification) of the fetal skeleton (by 0 - 0.5 days). No skeletal malformations or variations were reported.

Dose descriptor:

NOAEL

Effect level:

5 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Read-across justifications and data matrices are presented in IUCLID section 13.

Conclusions:

Ethanol was added to the diets of female rats for six weeks (from 3 weeks prior to mating to gestational day (GD) 21) in an attempt to determine effects on fetal growth and skeletal development. Based on the study results developmental toxicity NOAEL of 5200mg/kg/bw was established.

Executive summary:

Groups of female Sprague-Dawley rats were given liquid diets with 15, 25, or 36% ethanol-derived calories (E15, E25, and E36 groups, respectively), or without ethanol (pair-fed isocaloric (PF15, PF25, PF36) or ad libitum (C) control) for 3 weeks prior to mating, and throughout 21 days of gestation.

 

Prenatal ethanol exposure at 36% ethanol-derived calories (E36; about 10.4 g ethanol/kg bw/day) decreased fetal body weight and length, and skeletal ossification (a developmental delay), compared with pair-fed (PF36) and ad libitum controls at GD 21. Significant effects on ossification, but not body weight or length, were seen at E25 (corresponding to a dose of about 8.2 g ethanol/kg bw/day), compared to PF25 isocaloric controls. The NOAEL for this study can be considered to be 5.2 g ethanol/kg bw/day, as at this dose level (E15), no significant effects on fetal growth or ossification were seen compared to the PF15 isocaloric controls. A delay in the development of body weight and skeletal ossification was seen in the ethanol-treated (E25 and E36) fetuses on GD21, compared to the ab libitum controls. There were no skeletal malformations or variations (other than the delay in ossification) reported for any of the ethanol-treated groups.

 

The data indicate that ethanol has differential effects on fetal weight and skeletal development, and that the skeletal sites differ in their sensitivity to ethanol.

Reference 2

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A published study which contains sufficient experimental detail to judge results reliable. Read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture complete hydrolysis will take place with no significant reaction products other than alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the toxicokinetics of target substance. Because of the rapid hydrolysis, the influence of the mode of administration through inhalation, dermal and oral is related to the hazardous degradation product (alcohol) released from the target substance. The identification of degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance, which might change the hazardous properties of the target substance compared to the properties of the pure alcohol. As there is a mechanistic reasoning to the read-across, the unnecessary animal testing is avoided by using the read-across data from the degradation product (relevant alcohol) to evaluate irritation, sensitization and the short term and long-term toxicological effects and mutagenicity of the target substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

low number of pregnant females, no detailed examination of dams

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: not stated
- Weight at study initiation: 200-300g
- Fasting period before study: no
- Housing: 3 per cage in stainless-steel cages except whilst in chamber
- Diet (e.g. ad libitum): purina or comparable-grade lab chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:1-2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24
- Humidity (%): 20-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):12/12


IN-LIFE DATES: From: To:

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air in chamber

Details on exposure:

0.5m3 Hinner-type exposure chambers under negative pressure. Controls were placed in similar cage as the exposed animals with adjacent exposure chamber for the same hours.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Two methods used: Continuously by a Miran 1A general purpose infrared analyzer (Wilkes/Foxboro Analytical), on an hourly basis; and concentration samples taken from chamber atmosphere by charcoal tube. Sampling times 10-30 mins. 5-10 samples/week. Analysed by NIOSH 1977b-No. S-56 Method with slight modifications.

Details on mating procedure:

Virgin females were housed with males and vaginal smears were taken.

Duration of treatment / exposure:

7 hours per day in exposure chamber on gestation days 1-19. Animals left in the chambers for degassing for approximately 1/2 hr after vapor generation terminated.

Frequency of treatment:

daily

No. of animals per sex per dose:

not explicitly stated but approximately 16.

Control animals:

not specified

Details on study design:

Sex: female
Duration of test: Days 1-19 of gestation

Maternal examinations:

Blood levels

Ovaries and uterine content:

No maternal organs were examined

Fetal examinations:

Foetuses were examined externally and internally for malformations; implants and resorptions were recorded as was litter weight.

Statistics:

Kruskal-Wallis analysis of variance or Fisher's test.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No mortality occurred. Food consumption was lowered in the high-dose group. Clinical signs observed included complete narcosis (described as severe toxicity) in the highest dose induced. The lower doses did not cause narcosis but caused hyperactivity after exposure. Maternal weight gains were not affected by treatment. Blood alcohol levels ranged from 0.02 to 0.03 mg/ml at 10000 ppm, 0.42 to 0.84 mg/ml at 16000ppm and 1.48 to 1.93 mg/ml at 20000 ppm. Measurements were made on non-pregnant rats and represent the ranges of the average values measured at days 1, 10 and 19.

Dose descriptor:

NOAEL

Effect level:

16 000 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

20 000 ppm

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of pregnant per dose level were 15/15, 15/15, 15/16 and 14/16 in the control, low, medium and high dosage groups. The number of resorptions were not affected by ethanol inhalation. The number of implantations were 14-16/litter in all ethanol-treated groups and 15/litter in the control group. The number of corpora lutea were 14-16/litter. LItter weights were not significantly affected by ethanol treatments. The sex ratio did not differ significantly from controls. Grossly visible abnormalities are given in detail but the frequency of each did not differ significantly between groups. More litters contained abnormal foetuses in the 20,000 ppm group compared to controls but differences were only of borderline statistical significance.

Dose descriptor:

NOAEL

Effect level:

>= 20 000 ppm

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Maternal LOAEL effect was narcosis and lowered food consumption.

Development LOAEL effect – none seen

Development NOAEL effect – visceral or skeletal malformations or variants.

Maternal data are not given for the following: Number aborting; (Duration of pregnancy not relevant); bodyweights; haematology and blood chemistry findings; gross pathology in dams; organ weight changes; histopathology incidence and severity.

Foetal data are not given for the following: litter size; number viable; postnatal growth (not applicable) and postnatal survival (not applicable).

Abstract of article refers to maternal weight data that is not provided within results.

Conclusions:

Pregnant female rats were exposed to ethanol by inhalation at concentrations of 10000, 16000, or 20000ppm on gestation days 1 -19. Based on the study results the following NOAELs were established: NOAEL (maternal toxicity) :16,000ppm (30,400mg/m3) and NOAEL (teratogenicity): 20,000ppm (38,000mg/m3).

Executive summary:

Pregnant female rats were exposed to ethanol by inhalation at concentrations of 10000, 16000, or 20000ppm in a chamber for 7 hours per day on gestation days 1 -19. On day 20 the animals were euthanized and their fetuses examined. There was no definite increase in malformations at any level of ethanol exposure, although the incidence in the 20000ppm group was of borderline significance. There was clear maternal toxicity evident at the highest dose (narcosis, food intake reduction).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

304 000 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Testing is not necessary since there is enough evidence on the effects of the main decomposition products on development to conclude that titanium (4 +) ethanolate is not toxic to reproduction. As the substance hydrolyses rapidly (<5min) when it comes in contact with water or moisture (Brekelmans, M.J.C., 2013), intrinsic properties are related to the degradation products. After hydrolysis no significant reaction products other than ethanol and non-hazardous hydrated titanium dioxide exist.

Ethanol has harmonized classification according to EU regulation No. 1272/2008 (CLP) and it is not classified as toxic to reproduction. There are several studies available for ethanol to evaluate the developmental toxicity of titanium (4 +) ethanolate. The other decomposition product of titanium (4+) ethanolate is non-hazardous TiO2. Thus, it is concluded that there is no relevance to further evaluate TiO2 in the chemical safety assessment.

In the following sections studies related to developmental toxicity of ethanol are discussed in more detail.

Pregnant female rats were exposed to ethanol by inhalation at concentrations of 10000, 16000, or 20000 ppm in a chamber for 7 hours per day on gestation days 1 -19 (Nelson et al., 1985). On day 20 the animals were euthanized and their fetuses examined. There was no definite increase in malformations at any level of ethanol exposure, although the incidence in the 20000 ppm group was of borderline significance. There was clear maternal toxicity evident at the highest dose (narcosis, food intake reduction). A NOAEL for maternal toxicity of 16 000 ppm (30 400mg/m3) and a NOAEL for teratogenicity of 20 000 ppm (38 000mg/m3) was established for ethanol.

Groups of female Sprague-Dawley rats were given liquid diets with 15, 25, or 36% ethanol-derived calories (E15, E25, and E36 groups, respectively), or without ethanol (pair-fed isocaloric (PF15, PF25, PF36) or ad libitum control) for 3 weeks prior to mating, and throughout 21 days of gestation (Simpson, et al., 2005). Prenatal ethanol exposure at 36% ethanol-derived calories (E36; about 10.4 g ethanol/kg bw/day) decreased fetal body weight and length, and skeletal ossification (a developmental delay), compared with pair-fed (PF36) and ad libitum controls at GD 21. Significant effects on ossification, but not body weight or length, were seen at E25 (corresponding to a dose of about 8.2 g ethanol/kg bw/day), compared to PF25 isocaloric controls. The NOAEL for this study can be considered to be 5.2 g ethanol/kg bw/day, as at this dose level (E15), no significant effects on fetal growth or ossification were seen compared to the PF15 isocaloric controls. A delay in the development of body weight and skeletal ossification was seen in the ethanol-treated (E25 and E36) fetuses on GD21, compared to the ad libitum controls. There were no skeletal malformations or variations (other than the delay in ossification) reported for any of the ethanol-treated groups.

Overall, ethanol can elicit adverse effects on the reproductive system and on fertility in males and females and can trigger developmental toxicity in females. However, the ethanol doses required to cause developmental toxicity effects in animals are exceeding high compared to doses normally used to assess the hazards of chemical substances. It can be concluded that blood ethanol concentrations resulting from ethanol exposure at doses relevant to occupational exposure and the use of titanium (4+) ethanolate are unlikely to produce reproductive or developmental toxic effects.

Justification for selection of Effect on developmental toxicity: via oral route:
Unstable substance and therefore properties of developmental toxicity is related to degradation product (ethanol).

Justification for selection of Effect on developmental toxicity: via inhalation route:
No studies available for the target substance which is highly reactive. Data is obtained from the reliable study performed for the main decomposition product (ethanol).

Justification for selection of Effect on developmental toxicity: via dermal route:
Not likely route of exposure.

Justification for classification or non-classification

The weight of evidence on decomposition of this substance and the studies available from the main degradation products, ethanol and hydrated titanium dioxide, indicate that there is currently no need for classification of titanium (4 +) ethanolate concerning toxicity to reproduction or teratogenicity according to the CLP Regulation (EC) 1272/2008 and EU Directive 67/548/EEC.