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Key value for chemical safety assessment

Effects on fertility

Additional information

oral

A GLP conform repeated dose/ reproductive toxicity study was performed with TP-90B RUBBER CHEMICAL according to OECD 422 (RTC/ Rohm & Haas 2004). Groups of 10 Sprague-Dawley rats per sex received TP-90B Rubber Chemical in 0.5% aqueous carboxymethylcellulose at doses of 10, 100 and 800 mg/kg bw/d by gavage. Males were treated for 2 weeks prior to pairing, through pairing until the day before sacrifice. Females were treated for 2 weeks prior to pairing, through the mating and gestation periods up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum (= at least 43 days).

Following effects on parental toxicity were observed:

 

Fate of females

One mid dose female was sacrificed on Day 0 post-partum for human reasons since signs of dystocia (difficult delivery) were noted.

 

Weekly clinical signs and neurotoxicity assessment and daily pre- and post-dose observations

Weekly physical examinations including detailed clinical signs and neurotoxicity assessment (Functional Observation Battery) did not show any treatment-related effects.

Daily post-dose observations of clinical signs showed high dose male animals with ataxia,

Semiclosed eyes, hunched posture, reduced activity and salivation. Pronation, ataxia, semiclosed eyes, reduced activity, hunched posture, lethargy, twitches, piloerection and salivation were noted at post-dose observations in high dose females.

 

Body weight and body weight gain

Slight decreases in body weight and body weight gain occurred only in high dose females during gestation.

 

Food consumption

Food consumption remained comparable between control and treated groups in both sexes before pairing. Reduced food consumption was noted in high dose females on Days 7 and 14 post-coitum.

 

Motor activity and sensory reaction to stimuli

Motor activity measurements and sensory reaction to stimuli were unaffected by treatment at any dose level in both sexes.

 

Haematology

No toxicologically significant change in haematology values occurred in males or females at any dose level.

 

Clinical chemistry

No toxicologically significant change in any clinical chemistry parameter occurred in males or females at any dose level.

 

Terminal body weight and organ weights

No differences in terminal body weights were observed between treated and control animals.

Absolute and relative liver weights were increased in high dose males and females when compared to controls. All changes were statistically significantly different from controls except the absolute liver weight in females.

 

Macroscopic examination

No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test item. The gross changes observed were considered to be incidental or spontaneous in origin.

 

Microscopic examination

No microscopic changes clearly related to test item administration were observed in male or female rats in the low and mid-dose groups. Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dose group. A finding of uncertain significance was observed in the high dose group.

Focal germ cell depletion and degeneration in one testis (unilateral) occurred in four high dose male rats. Three of these four rats also had a few exfoliated spermatogenic cells in the lumen of the epididymal tubules.

 

 

Following effects on fertility were observed:

Reproductive parameters

Oestrus cycle, copulation plug, pre-coital interval and copulatory index at all dose levels were comparable to controls. Fertility index in the high dose group was decreased compared to control: Thirty-nine females in the study showed positive signs of mating. Fifteen females proved not to be pregnant at necropsy; three in the control and in the mid-dose group, two in the low dose group and seven in the high dose group. In addition, two control and one mid-dose female showed unilateral implantation. One low dose female did not mate. The number of females with live pups on Day 4 post-partum was 6, 8, 6, and 3 in the control, low, mid and high dose groups, respectively.

 

Implantation and pre-birth loss

Gestation periods were similar between control and treated groups. All dams gave birth on Day 22 or Day 23 post-coitum. The number and percentage of pre-birth loss of pups in the high dose females was increased compared to controls.

 

Litter data and sex ratios

Cumulative pup loss was statistically significantly increased in the high dose group on Day 4 post-partum. Litter weight and mean pup weight in the high dose group were lower than controls at birth and on Day 4 post-partum. A decrease in the average number of pups per litter was observed at birth and on Day 4 post-partum in the high dose group. Pup survival to Day 4 post-partum was decreased in the high dose group. Increased pup mortality from Day 0 to Day 4 was noted in the high dose group. Sex ratios at birth and on Day 4 post-partum did not show any differences between control and treated groups.

 

 

Based on the results of the study, the NOAEL for maternal toxicity is considered to be 100 mg/kg bw/day due to decreased body weight, food consumption, increased liver weights and microscopic liver changes; the NOAEL for effects on reproduction is considered to be 100 mg/kg be/day due to decreased fertility indices and embryo-fetal toxicity. However, a low fertility rate was observed also in the control group and historical control data useful for assessment are missing in the report. Additionally, adverse effects were observed only in the highest dose without a clear dose relationship. In conclusion, a final assessment of the observed effects cannot be made.

Summary of the concerns with the reliability of this study:

The relevance of the observed effects (decreased fertility indices and increased pre-birth losses,

embryo-fetal toxicity as well as developmental effects of microdactyly and/or agenesis of digits (one litter) and

flexure of hind limbs (one litter)) with regard to the hazard assessment

(i.e., classification) is unclear due to the following concerns about the study:

 Low fertility rate for the control group which limits the predictive (i.e., statistical)

power of the study as well as its reliability

 Lack of a clear dose-response relationship (effects only in the high dose group);

Observed parental toxicity raises the concern that 800 mg/kg for the high dose level

may have been too high

 Inadequate sample size for evaluation of fetal malformations in the high dose

group

 Lack of the historical control data in the study report does not allow for

distinguishing between substance-related and spontaneous effects

 Some incorrect values stated in the final report call into question the QA process

for the study, the data collection software, and /or perhaps some of the results


Short description of key information:
oral
Rat, Combined repeated dose toxicity/ Reproduction toxicity screenig study: NOAEL parental toxicity = 100 mg/kg bw due to decreased body weight, food consumption, increased liver weights and microscopic liver changes in the higher dose; NOAEL fertility = 100 mg/kg bw due to decreased fertility indices and increased pre-birth loss (GLP, OECD 422, RTC/ Rohm & Haas 2004)

Effects on developmental toxicity

Description of key information
OECD 414 (RTC, 2013):  The mid-dose level (120 mg/kg bw/day) was considered the NOAEL (No Observed Adverse Effect Level) for maternal toxicity.
The NOAEL for developmental toxicity and teratogenicty was considered to be 400 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Additional information

oral

A study was conducted (RTC, 2013) according to OECD 414 guideline and GLP. The study investigated the effects of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane on pregnancy and embryo-foetal development in the Sprague Dawley rat following oral administration, according to the following experimental design:

 

Group

Treatment

Number of

Number

(mg/kg/day)+

Animals

1

2

3

4

0

40

120

400

24 F

24 F

24 F

24 F

+: in terms of test item as supplied

F: females

 

Females were dosed starting from Day 6 until Day 19 post coitum.

The test item was administered, by the oral route, at a dose volume of 10 mL/kg.

The vehicle was an aqueous solution of carboxymethylcellulose (CMC 0.5% w/v).

The following investigations were performed: mortality check, clinical signs, body weight, food consumption and macroscopic observation of the females.

On Day 20 post coitum at necropsy, gravid uterus, corrected body weight (terminal body weight minus gravid uterus weight), corrected body weight gain (terminal body weight minus body weight at Day 6 post coitum minus gravid uterus weight), corpora lutea, implantation sites, number, sex and weight of all live foetuses were determined. Gross evaluation of the placenta was also performed.

 

Mortality and fate of females

No mortality of females occurred during the study.

One control female and one mid-dose female were found not pregnant at necropsy.

The number of females with live foetuses on Day 20 post coitum was 23 in the control group, 24 in the low dose group (40 mg/kg/day), 23 in the mid-dose group (120 mg/kg/day) and 24 in the high dose group (400 mg/kg/day).

 

Clinical signs

Treatment-related clinical signs noted in females receiving 400 mg/kg bw/day were ataxia, salivation and lethargic aspect. Other clinical signs, such as tremors, bradypnoea and piloerection were also noted.

One female at 120 mg/kg bw/day showed prone position and salivation on one day.

Females receiving 40 mg/kg/day did not show any clinical signs.

 

Body weight and body weight gain of females

A statistically significant decrease in body weight gain (approximately 35%) was observed on Day 9 post coitum in treated females receiving 400 mg/kg bw/day.

 

Food consumption

A slight decrease on food consumption was noted in females receiving 400 mg/kg bw/day on Day 9 post coitum.

 

Terminal body weight, uterus weight, corrected body weight and corrected body weight gain of females

 

A decrease in body weight gain from Day 6 post coitum and Day 20 post coitum corrected for gravid uterus weight was recorded in treated females receiving 400 mg/kg bw/day (approximately 20%).

 

Litter data and sex ratios

Litter data, mean foetal weight and sex ratios were not affected by treatment.

 

Macroscopic observations of females

No macroscopic observations were observed in females sacrificed on Day 20 post coitum on control and treated groups.

 

External examination of foetuses

A total of 6 small foetuses (foetal weight < 2.7 g) were detected: 1 out of 341 in the control group, 3 out of 357 in females receiving 40 mg/kg bw/day, 2 out of 345 in females receiving 120 mg/kg bw/day. One dead foetus was observed in a low dose litter (40 mg/kg bw/day) and a foetus showing cranioschisis was noted in another low dose litter. This malformation was considered incidental.

 

Fixed visceral examination of foetuses

No findings that could be considered treatment-related were noted at visceral examination of the foetuses in any group.

 

Skeletal examination of foetuses

Most of the alterations recorded at skeletal examinations were noted both in control and treated groups, with a similar incidence.

The foetus in the low dose group showing cranioschisis (see above) showed multiple malformations at skeletal examination. Unossified pubis was observed in another low dose foetus of a different litter.Considering that these alterations were observed in small foetuses (foetal weight<2.7 g), without dose-relation, they were considered incidental and not related to treatment.

 

Conclusions

On the basis of the results obtained in this study, Bis-(2-(2-butoxyethoxy)-ethoxy)-methane induced maternal toxicity at the dose level of 400 mg/kg bw/day, as shown by clinical signs (e.g., ataxia, tremors, etc.), and decreased body weight gain. Clinical signs at
120 mg/kg bw/day occurred in one animal only and were confined to salivation (Day 8 post coitum) and prone position (Day 10 post coitum) noted on one day. Due to the isolated and transient occurrence, these findings were not considered to be toxicologically relevant.

 

Therefore, the mid-dose level (120 mg/kg bw/day) was considered the NOAEL (No Observed Adverse Effect Level) for maternal toxicity.

The NOAEL for developmental toxicity and teratogenicity was considered to be 400 mg/kg bw/day.

Range-Finder for OECD 414 study:

The purpose of this study was to investigate the effects of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane on pregnant rats, following oral administration in order to select the dosages for the definitive prenatal developmental toxicity study. The dose levels were 0, 250 and 750 mg/kg bw/day. Females were dosed starting from Day 6 until Day 19 post coitum. The test item was administered, by the oral route, at a dose volume of 10 mL/kg and the vehicle was an aqueous solution of 0.5 % carboxymethylcellulose (0.5 % CMC).

Mortality and fate of females

No mortality occurred during the study.

All females in control and treated groups were found pregnant at necropsy.

Clinical signs

Treatment-related clinical signs noted in females receiving 750 mg/kg bw/day were ataxia, salivation and piloerection. Other clinical signs, such as decreased activity, lethargy and prone position occurred in these females. Sometimes dyspnoea or tachypnoea were also observed.

Females receiving 250 mg/kg bw/day showed salivation, while ataxia, decreased activity and lethargy were recorded sporadically.

Body weight and body weight gain of females

A slight and statistically significant decrease in body weight was noted in females dosed at 750 mg/kg bw/day, starting from Day 15 post coitum until Day 20 post coitum.

Statistically significant decrease in body weight gain was observed on Day 9 post coitum in treated females both at 250 and 750 mg/kg bw/day.

Food consumption

A decrease in food consumption was noted in females receiving 750 mg/kg bw/day on Days 9 and 12 post coitum.

Terminal body weight, uterus weight, corrected body weight and corrected body weight gain of females

Body weight and corrected body weight gain during gestation Day 6 to 20 of treated females showed a dose-depended decrease, even though not statistically significant.

Macroscopic observations of females

Macroscopic observations in females sacrificed on Day 20 post coitum did not show any evidence of treatment-related effects, when compared to the control group.

Conclusions

On the basis of the results obtained in this study, it could be concluded that Bis-(2-(2 - butoxyethoxy)-ethoxy)-methane induces slight to moderate signs of maternal toxicity at dose levels of 250 and 750 mg/kg bw/day, respectively.

Therefore, the high dose level for the subsequent main study should be lower than 750 mg/kg bw/day.

A GLP conform repeated dose/ reproductive toxicity study was performed with TP-90B RUBBER CHEMICAL according to OECD 422 (RTC/ Rohm & Haas 2004). The test methods, effects on maternal and fetal toxicity, and critical recommendations to the observed effects are already given above.

Following effects on developmental toxicity/ teratogenicity were observed:

Pre-weaning clinical signs and necropsy findings of F1 pups

Six pups in one litter of the high dose group showed forehind digits missing or not defined during pre-weaning observations and/or necropsy. Necropsy examination of four of these pups (two additional pups were not available for necropsy) confirmed the diagnoses of agenesis and/or microdactily. One pup from a different litter had flexure of the hindlimbs.

Based on the results of the study, the NOAEL for maternal toxicity is considered to be 100 mg/kg bw/day due to decreased body weight, food consumption, increased liver weights and microscopic liver changes; the NOAEL for teratogenic effects is considered to be 100 mg/kg be/day due to observed malformations of the extremities of high dose pups. However, individual data for gross fetal examinations and helpful historical control data are missing in the report and the sample size (number of pups) is inadequate for statistical evaluations of fetal malformations as only 3 rats delivered pups. Additionally, adverse effects were observed only in the highest dose without a clear dose relationship. In conclusion, a final assessment of the observed effects cannot be made.

Summary of the concerns with the reliability of this study:

The relevance of the observed effects (decreased fertility indices and increased pre-birth losses,

embryo-fetal toxicity as well as developmental effects of microdactyly and/or agenesis of digits (one litter) and

flexure of hind limbs (one litter)) with regard to the hazard assessment

(i.e., classification) is unclear due to the following concerns about the study:

 Low fertility rate for the control group which limits the predictive (i.e., statistical)

power of the study as well as its reliability

 Lack of a clear dose-response relationship (effects only in the high dose group);

Observed parental toxicity raises the concern that 800 mg/kg for the high dose level

may have been too high

 Inadequate sample size for evaluation of fetal malformations in the high dose

group

 Lack of the historical control data in the study report does not allow for

distinguishing between substance-related and spontaneous effects

 Some incorrect values stated in the final report call into question the QA process

for the study, the data collection software, and /or perhaps some of the results

Justification for classification or non-classification

Based on the available data on the reproductive toxicity of hexaoxatricosane no classification is warranted according to the criteria of both, 67/548/EEC and CLP regulation (EC) 1272/2008.