tert-butyl hydroperoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 422 study is available on TBHP with the NOAEL detailed as the highest dose (21 mg/kg/day). The requirement for an EOGRTs is fulfilled using read-across to the source substance ETBE where a two generation reproductive toxicity study is available. This study provided a NOAEL value of 1000 mg/kg/day for reproductive effects (no adverse effects at the highest dose tested).

Full details and justifications on the read-across are included in the appropriate target substance record in section 7.8.1 of IUCLID.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, acceptable for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatstoezicht op de Volkgezondheid, 19 April 1989; 6 June 1991

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wigd GmbH, Sulzfeld, Germany
- Age at study initiation: approx. 9-10 wk
- Weight at study initiation: male = 311.1 g; female = 198.5 g
- Housing: individually (during premating, gestation and lactation) or in pairs of one male and one female (during mating)
- Diet (e.g. ad libitum): cereal-based stock diet ad libitum
- Water (e.g. ad libitum): tap-water ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Solutions of test substance were prepared in Millipore-filtered water and stored in brown glass bottles, in a refrigerator, until use.

Concentration in dosing solutions (mg Aq. TBHP-70/ml):
Main study: 0, 0.3, 1, 3

Dosing volume: 10 ml/kg bwt

The test substance was administered once daily by oral gavage (10 ml/kg) with the dose volume were adjusted for changes in body weight. Four batches of dosing solutions were prepared for the main study (on June 30, July 14 and 28, and August 11, 1992).
The main study commenced on 1 July 1992 (= nominal day 0) and was terminated on 10-14 August 1992 and the animals sacrificed a few hours after the last dose.

Animals received treatment for up to 45 consecutive days.

Details on mating procedure:

Following a 2 wk premating treatment period, one male was paired with one female of the same treatment in the male home cage. Females were checked the following morning for evidence of mating (either the presence of a vaginal plug or the presence of sperm in a vaginal smear). Females that had not mated were rehoused with the same male and the process repeated for up to one week. The day of detection of a vaginal plug or sperm in vaginal lavage was designated day 0 of pregnancy. Females were returned to their home cage after mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were analysed by GC-FID using a Perkin Elmer 8500 gas chromatograph with fused silica 50 m x 0.21 mm column and 0.5 um film methylsilicon PONA. Stability was determined at room temperature for 3 days or in a refrigerator for up to 16 days. Homogeneity was checked by analysing samples from the top, middle and bottom of the dosing solutions. Results demonstrated that achieved concentrations were nominal.

Duration of treatment / exposure:

Main study: 41-45 days (in-life phase 1 July 1992 to 10-14 August 1992)

Frequency of treatment:

Daily

Details on study schedule:

Treatment commenced: 1 July 1992
Mating commenced: 15 July 1992
In-life phase ended: 14 August 1992

Remarks:

Doses / Concentrations:
0, 3, 10 or 30 mg/kg bwt/d
Basis:
nominal in water

Remarks:

Doses / Concentrations:
2.1, 7 or 21 mg/kg bwt/d
Basis:
other: actual dose received

No. of animals per sex per dose:

12 males, 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance was administered once daily by oral gavage for up to 45 consecutive days.

Positive control:

not relevant

Parental animals: Observations and examinations:

See Chapter 7.5.1, record Repeated dose toxicity: oral.001

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

The following observations were recorded on PND1 and PND4:
- litter size, number of live and dead pups
- liver litter weight
- number of males and females
- number with external abnormalities

Postmortem examinations (parental animals):

Necropsy and histopathology procedures are described in Chapter 7.5.1, record Repeated dose toxicity: oral.001

Postmortem examinations (offspring):

Surviving pups were sacrificed (ether) on PND4 and subject to macroscopic examination

Statistics:

Pregnancy, litter and survival data along with clinical and macroscopic findings were analysed using Fisher's exact probability test. Time to mating, duration of gestation and litter size were analysed by Kruskal-Wallis ANOVA followed by the Mann-Whitney U-test, and pup body weights by ANOVA followed by Dunnett's multiple comparison tests.

Reproductive indices:

The following reproductive indices were calculated:
- mating index
- fertility index
- fecundity index
- gestation index
- sex ratio

Offspring viability indices:

The following offspring viability indices were calculated:
- live birth index
- viability index

Reproductive performance:

no effects observed

Gross pathology and histopathology
Results from range-finding studies preliminary to this investigation revealed the presence of moderate to severe submucosal oedema in the stomach wall of rats given doses of 107 mg/kg bwt/d for 5 consecutive days; no effects at 30 mg/kg bwt/d. Other information regarding findings in F0 parental animals from this study is reported in Chapter 7.5.1.

Reproductive performance
There were no treatment-related differences in:
- precoital time
- the number of pregnant females
- duration of gestation
- mean numbers of corpora lutae
- number of total, pre-implantation and post-implantation sites

Female fertility and fecundity indices were unexpectedly low in the control, low and high dose groups. Re-examination of vaginal smears from these animals revealed no false-positives and the cause of these findings (considered unrelated to treatment by the study director) is unclear:

Selected results by dose level (0, 3, 10 or 30 mg/kg bwt/d):
- females mated: 11/12, 11/12, 11/11, 11/12
- females pregnant: 8/11, 5/11, 10/11. 7/11
- females with live pups: 8/8, 4/5, 10/10, 7/7
- mating index (%): 92, 92, 100, 92
- female fertility index (%): 67, 42, 91, 58
- female fecundity index (%): 73, 45, 91, 64
- gestation index (%): 100,100, 100, 100

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no adverse effects at highest dose tested

Basis for effect level:

other: Remark: toxicokinetic data demonstrate rapid conversion of TBHP to 2-methylpropan-2-ol (tert-butanol) in vivo, thereby minimising any potential for uptake and systemic distribution of unchanged TBHP following oral exposure.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified Generation not specified (migrated information)

Mortality / viability:

no mortality observed

The mean number of pups per litter and sex ratio was comparable in all groups, however pup mortality between PND1 and PND4 appeared significantly increased in low and high dose litters but not in the intermediate dose group (i.e. no trend present):

Results by dose level (0, 3, 10 or 30 mg/kg bwt/d):
- mean pups/litter: 11,00, 10.75, 11.10, 11.57
- live birth index (%): 98.9, 100, 100, 100
- pup mortality day 1 (%): 1.1, 0.0, 0.0, 0.0
- pup mortality day 4 (%): 0.0, 26 ***, 0.9, 6.2*
- viability index day 1-4 (%): 100, 74, 99, 94

*p<0.05; *** p<0.001

Comment: The mortality recorded in the low dose group was strongly influenced by the loss of all 10 pups from one litter. This finding together with the absence of any dose-response relationship lead the study director to conclude that this was unrelated to treatment.
Comment: The finding of a significant increase in pup mortality in the high dose group (reflecting the death of 1 or 2 pups in 4 of 7 litters) was considered by the study director to be an artefact caused by the high survival of the control litters.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no adverse effects at highest dose tested

Reproductive effects observed:

not specified

The relevance of mortality present in the low and high dose groups from this study was considered in the TBHP risk assessment report (document R319_0710_HH dated November 2007; Chemical Substances Bureau, Bilthoven, the Netherlands), which noted:

No significant toxicological effects were observed on reproductive indices and litter data, except for pup mortality between day 1 and 4 post partum in the 3 and 30 mg/kg bw groups (control: 0/87 (0%), 3 mg/kg: 11/43 (26%) with one total litter loss of 10 pups, 10 mg/kg: 1/111 (1%) and 30 mg/kg: 5/81 (6%). The study authors stated that the loss of pups in the 3 mg/kg bw group was probably not toxicological relevant because these pups were largely from one litter, and because no increased mortality in the next higher dose group was observed. The higher pup mortality at 30 mg/kg could be treatment related according to the study authors. However, the incidence of postnatal death was considered to be very low and therefore of minor toxicological significance. No information on historic control data were provided to show that a 6% postnatal mortality is within the normal range. No toxicological relevant effects on body weight, clinical observations and macroscopical observations in the pups were observed. From these results it can be concluded that the NOAEL for reproductive/developmental toxicity in this study is 30 mg/kg bw 70% TBHP, the highest dose tested.

The findings were not considered sufficiently robust to support classification of TBHP as substance toxic to reproduction by the EU Classification and Labelling Work Group.

Conclusions:

No evidence of adverse effects on parental reproductive indices and litter data following repeated oral exposure.

Executive summary:

The reproductive toxicity of TBHP was investigated in a GLP-compliant guideline combined repeated dose and reproductive/developmental toxicity screening test (OECD 422)  in which groups of male and female Wistar rats (12/sex/dose level) received TBHP by oral gavage in water at nominal doses of 0, 3, 10 or 30 mg/kg bw/day for 45 consecutive days (actual received doses = 0, 2.1, 7 or 21 mg/kg bwt/day). Treatment commenced 2 weeks prior to mating. Dose levels were based on results obtained from two preliminary range-finding studies which demonstrated moderate to severe damage to the stomach lining at higher treatment levels. There were no significant treatment-related toxicological effects observed on parental reproductive indices and litter data, with a NOAEL of 21 mg TBHP /kg bwt/d (actual dose received).

Reference 2

Endpoint:

two-generation reproductive toxicity

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:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Standard as per guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: time-mated female Sprague Dawley rats (Charles River Laboratories, L'Arbresle, France)
Age at start of treatment: approx. 7 wk (mean bw 203 g males, 157 g females)
Acclimation period: 6 d
Housing: individually housed, stainless steel cages (wire mesh bottom) for duration of study
Diet: A04 C pelleted maintenance diet (UAR, Villemoisson, France), ad libitum
Water: deionised water, ad libitum
Assignment to treatment groups: computer-generated weight randomisation

The animal room conditions are set as follows:
- temperature: 22 ± 2°C
- relative humidity: 50 ± 20%
- light/dark cycle: 12h/12h (7:00 - 19:00)
- ventilation: about 12 cycles/hour of filtered, non-recycled air.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ETBE was administered to groups of 25 male and 25 female rats by gavage at doses of 0 (corn oil; 4 ml/kg bw), 250, 500 or 1000 mg/kg/day for 7 days/week as follows:

F0 generation:
- males: from 10 wk before mating, during 2 wk mating period and until sacrifice (after weaning of F1 pups), total duration of treatment: 18 weeks.
- females: from 10 wk before mating, during 2 wk mating period, pregnancy and lactation until day 21 post-partum, total duration of treatment: 18 weeks.

F1 and F2 generations:
- as above, commencing day 22 post-partum

Details on mating procedure:

Mating followed a 10 wk treatment period. Dosing of F0 animals began at age 7 wk, with mating from age approx. 17 wk. Dosing of F1 animals began at weaning when the animals were 22 d old, with mating from age approx. 13 wk.

Females were monitored for estrous stage (microscopic examination of fresh vaginal lavage, stained with methylene blue) each morning during the last 3 weeks of the pre-mating period and during the mating period (until mating confirmed by presence of a vaginal plug or sperm in a vaginal lavage).

Females were paired overnight with males from the same dose group until mating had occurred or 14 days had elapsed, whichever occurred first. Parental F1 animals were selected from different litters to avoid brother-sister matings.

Pregnant females were housed individually, allowed to litter normally and raise progeny until weaning.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions were prepared weekly (based upon stability data demonstrating no losses of ETBE over 9 d) and samples taken for analysis (GC) to confirm received dose. Stability and homogeneity studies returned satisfactory results (all analysed concentration within 10% of nominal).

Duration of treatment / exposure:

F0 generation:
- males: from 10 wk before mating, during 2 wk mating period and until sacrifice (after weaning of F1 pups)
- females: from 10 wk before mating, during 2 wk mating period, pregnancy and lactation until day 21 post-partum

F1 and F2 generations:
- as above, commencing day 22 post-partum

Frequency of treatment:

7 days/week

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose-levels were selected in agreement with the Sponsor on the basis of a previously conducted study (CIT/Study No. 24168 RSR). The results demonstrated a slight but statistically significant decrease in body weight for pregnant female rats given 1000 mg/kg/day, and ptyalism (excess salivation) over the course of the study in both sexes given 1000 mg/kg/day. No effect on mating, pregnancy, lactation or litter data parameters was noted at 50, 250, 500 and 1000 mg/kg/day in this preliminary investigation. Consequently, dose-levels of 250, 500 and 1000 mg/kg/day were selected for use in the main two-generation study. It is noted that 1000 mg/kg/day is considered a limit dose for this type of investigation (OECD Guideline 416).

Positive control:

Not a guideline requirement

Parental animals: Observations and examinations:

Parental animals were observed at least twice daily for clinical signs, morbidity or mortality. Body weight and food consumption was recorded weekly for males, and at least weekly for females, commencing during the pre-mating period.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined by microscopic examination of a fresh vaginal lavage (stained with methylene blue), each morning as follows:
- during the last 3 weeks of the pre-mating period;
- during the mating period, until mating was confirmed.

Sperm parameters (parental animals):

Sperm collected (under isoflurane anaesthesia, prior to terminal sacrifice) from F0 and F1 parental males for quantitative evaluation of:
- epididymal sperm motility (x40 magnification), sperm count (cauda sperm count, Malassez cell) and sperm morphology (eosin staining, x40 magnification)
- homogenisation resistant testicular sperm (Neubauer cell)

Litter observations:

Total litter size, number of live/dead pups and sex of each pup was recorded as soon as possible after birth, and the pups observed daily for clinical signs. Anogential distance was measured (both generations) on day 1. Any gross malformations were noted. The size of each litter was adjusted to 4 males and 4 females on post-partum day 4. Pup body weight was recorded on days 1, 4, 7, 14 and 21 post-partum.

Reflex development was recorded in F1 and F2 pups as follows:
- surface righting reflex on day 5 post-partum
- cliff avoidance on day 11 post-partum
- air-righting reflex on day 17 post-partum

Acoustic startle response and pupil constriction reflex were assessed in F1 pups at 4 wk of age. Spontaneous motor activity was evaluated in F1 pups twice, at weekly intervals, when the animals were 7-8 wk old.

Males monitored between 32-47 d old for preputial separation, females monitored between 28-40 d old for vaginal opening (pups from both generations).

Postmortem examinations (parental animals):

Parental animals sacrificed (carbon dioxide) after weaning of the litters (that is, between day 22-25 post-partum), weighed, and subject to a gross internal examination (including examination of the uterus for implantation scars).

Following organs weighed:
Males:
- testes (separately), epididymides (separately), prostate, seminal vesicles together with coagulating glands, brain, liver, kidneys, spleen, pituitary gland, thyroids with parathyroids, adrenals
Females:
- uterus, ovaries, brain, liver, kidneys, spleen, pituitary gland, thyroids with parathyroids, adrenals,

PRESERVATION OF TISSUES:

The following organs preserved from all F0 and F1 parental animals:
ovaries and oviduct,
uterus (with cervix and horns)
vagina
testis (right)
epididymis (right)
seminal vesicles
prostate
coagulating glands
pituitary gland
adrenal gland
A vaginal smear taken from all females and stained (Harris Schorr staining).
Any abnormal tissue found at necropsy in the weanling pups was preserved.

EXAMINATION OF TISSUES

The following tissues from control and high dose F0 and F1 animals stained with haematoxylin-eosin prior to microscopic
examination:
any macroscopic abnormalities
ovaries and oviducts (including qualitative assessment of
primordial follicle population)
uterus (with cervix and horns)
vagina
epididymis
seminal vesicles
prostate
coagulating glands
pituitary gland
adrenal gland
The vaginal smear taken from females at terminal sacrifice also examined microscopically.

The testis from all treated males plus the controls was stained with haematoxylin/PAS and subject to a detailed histopathological examination (including an assessment of retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells).
Any macroscopic abnormalities present in the pups born to F0 and F1 mothers were prepared for histopathological evaluation.

Postmortem examinations (offspring):

Three pups/sex/litter born to the F0 and F1 mothers subject to gross necropsy at weaning. Brain, spleen and thymus removed and weighed.

Statistics:

Mean values were compared by one-way ANOVA and Dunett's test (mean values being considered as normally distributed and variances being considered homogenous). Percentage values were compared by the Fisher exact probability test.
Mean percentages of motility and morphology sperm parameters were Arcsine transformed and then compared by ANOVA. Mean values for anogenital distance, ratio of anogenital distance to pup body weight and anogenital distance normalized to the cube root of the individual pup's body weight were compared by analysis of variance.

Reproductive indices:

F0 generation:

Mating indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).
Fertility indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).

F1 GENERATION

Mating indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).
Fertility indices for males and females comparable among the groups and unaffected by treatment (no trend or statistically significant differences present).

Offspring viability indices:

F0 litters/F1 pups:
0 250 500 1000 mg/kg/d
Viability index on LD4 (%) 97.6 92.9 82.3 97.7

F1 litters/F2 pups:
0 250 500 1000 mg/kg/d
Viability index on LD4 (%) 97.6 94.8 97.0 92.9

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

PARENTAL/ADULT OBSERVATIONS:
F0 PARENTAL GENERATION
No deaths in either sex during the pre-mating, mating or gestation periods. One female given 250 mg/kg bw/d and three females given 500 mg/kg bw/d sacrificed during the first week of lactation because their entire litters died or were in poor condition. Considered unrelated to treatment by studydirector since no comparable findings in the high dose group.
Ptyalism (excessive salivation) observed in all treated groups (dose-related trend), considered related to unpalatable nature of the dosing solutions by study director.
Body weight gain decreased significantly in mid (-29%; P<0.01) and high (-22%; P<0.001) males on study days 85-113 (i.e. final 28 days of treatment, following mating). Considered treatment-related by study director. Female body weights unremarkable. Food intake not adversely affected by treatment in either sex.

F1 PARENTAL GENERATION
One control male sacrificed due to poor condition on study day 36, one female given 1000 mg/kg bw/d found dead on day 47, and one low dose female and one high dose female sacrificed early because their entire litters were found dead a few days after birth. These findings considered unrelated to treatment by the study director.
Ptyalism (excessive salivation) observed in all treated groups (dose-related trend), considered related to the unpalatable nature of the dosing solutions by study director.
Body weight and food consumption unaffected by treatment in both sexes.

REPRODUCTIVE DATA FOR PARENTAL GENERATIONS:
F0 PARENTAL GENERATION
The majority of pregnancy and parturition parameters unremarkable, with implantation, fecundity, neo-natal loss, gestation and delivery unaffected by treatment. The exception was a slightly greater post-implantation loss in the low and mid dose groups, reflecting litters found dead or sacrificed prematurely due to poor clinical condition. Considered unrelated to treatment by study director since no similar findings in high dose group.

F1 PARENTAL GENERATION
Pregnancy and parturition parameters unremarkable, with implantation, fecundity, post-implantation loss, neo-natal loss, gestation and delivery unaffected by treatment. A single pregnant female from the 250 mg/kg bw/d group did not deliver, and was found to have only a single implantation site at necropsy. Considered unrelated to treatment by study director since only one animal from the low dose group affected.

SPERM ANALYSES FOR F0 AND F1 PARENTAL MALES
No statistically significant or dose-related changes in seminology data for F0 and F1 males. A slightly lower testicular sperm head count noted in all treated F0 males appeared to be the consequence of a high control value. Overall, sperm parameters unaffected by treatment in both generations at all dose-levels.
------ mg/kg bw/d ------
0 250 500 1000
F0 MALES
Epididymal sperm
Spermatozoa (10^3/mm^3) 923 938 938 918
Motility (%) 99.7 100 98.6 97.6
Normal morphology (%) 93 93 97 96

Testicular sperm
Sperm heads (10^6/g testis) 148 109 108 110
Sperm production (10^6/g/d) 18.8 17.9 17.1 18.0

F1 MALES
Epididymal sperm
Spermatozoa (10^3/mm^3) 725 673 701 688
Motility (%) 84.6 87.1 93.3 88.3
Normal morphology (%) 84 86 86 88

Testicular sperm
Sperm heads (10^6/g testis) 100.6 97.8 105.3 99.8
Sperm production (10^6/g/d) 16.5 16.0 17.3 16.4

NECROPSY AND HISTOPATHOLOGY
F0 GENERATION PARENTS
Absolute and relative liver weights significantly increased in high dose males, and correlated with liver enlargement noted during necropsy and hepatocellular hypertrophy (considered adaptive response to treatment by the study director) in livers from three animals selected for histopathological examination. Liver weights from females given 1000 mg/kg/day slightly (increased 4-6%) but non-significantly increased. Kidney weights significantly increased in high dose males and correlated with the presence of microscopic acidophilic globules in cortical tubular epithelium from 5 of 6 animals selected for microscopic examination (considered related to alpha2u-globulin accumulation by study director). Kidney weights also significantly increased in male rats given 500 mg/kg/day (no microscopic examination performed).

Summary table: percentage change in F0 liver and kidney weights
--- Males --- -- Females --
250 500 1000 250 500 1000
Liver
- absolute +2 +2 +17** -1 +4 +6
- relative +3 +6 +24** +10 +8 +4

Kidney
- absolute +11 +15** +21** -1 +2 +5
- relative +11 +18** +28** +9 +5 +3

All other organ weight and macroscopic findings consistent with those observed in untreated rats of this strain and age, and considered of no toxicological importance by the study director.

F1 GENERATION PARENTS
Absolute and relative liver weights significantly greater in males given 500 or 1000 mg/kg/day and correlated with liver enlargement recorded at necropsy. Microscopic examination of liver tissue from two selected high dose males revealed slight or moderate hepatocellular hypertrophy, considered an adaptive response to treatment by the study director. Absolute and relative kidney weights significantly increased in males given 1000 or 500 mg/kg/day, with slight to marked accumulation of acidophilic globules (considered related to alpha2u-globulin accumulation by study director) detected microscopically in tissue from one selected mid dose male and four selected high dose males.

Summary table: percentage change in F1 liver and kidney weights
--- Males --- -- Females --
250 500 1000 250 500 1000
Liver
- absolute +0 +14** +27** +1 +3 +10**
- relative +0 +11** +25** +3 +6 +9*

Kidney
- absolute +10 +22** +58** +4 +3 +11**
- relative +10** +19** +58** +6 +6 +10**

All other organ weight and macroscopic findings consistent with those observed in untreated rats of this strain and age, and considered of no toxicological importance by the study director.

MICROSCOPIC EXAMINATION OF REPRODUCTIVE ORGANS
F0 GENERATION
Minimal degeneration of seminiferous tubules present in two control and two high dose animals, with desquamated spermatocytes present in control (8/25 animals) and treated high dose animals (1/25 animals). These findings commonly recorded in rats of this strain and age and considered of no
toxicological importance by the study director. Interstitial mononuclear cell aggregation and subacute prostatitis of the prostate and spermatic granuloma in the epididymides occurred with equal incidence and severity in both control and treated animals in this study, and considered of no toxicological importance by the study director.
Microscopic examination of the ovaries, uterus and vagina fromcontrol and high dose females revealed morphological changes consistent with a regular estrous cycle in both control and treated animals. Quantitative analysis of the primordial and growing follicles in the ovaries showed no perceptible differences between control and treated animals.

F1 GENERATION
A reduced number of tailed and round spermatids, spermatocytes, and spermatogonia present in 3/25 low dose males, 1/24 mid dose males and 3/25 high males versus 0/25 in the controls. In the majority of instances, changes correlated with seminiferous tubules lined with Sertoli cells only (minimal severity), with oligospermia or aspermia in the epididymides of the males receiving 1000 mg/kg/day (epididymides not examined in the low and intermediate dose-level groups). Minimal focal degeneration of germinal epithelium observed in 1/24, 1/25 or 1/25 males from the control, low- or high-dose groups, respectively. Minimally or slightly degenerated/necrotic cells observed in the lumen of the testis from 9/24 control males, 4/25 low dose males, 8/24 mid dose males and in 6/25 high dose males. Although some of these observations not present in the controls, the study director considered there was no evidence of a dose-response relationship in their incidence and/or severity, and that similar findings can occur spontaneously in the untreated rats of this strain and age. Consequently, findings considered unrelated to treatment.
Microscopic examination of the ovaries, uterus and vagina from control and high dose females revealed morphological changes consistent with a regular estrous cycle in both control and treated animals. Quantitative analysis of the primordial and growing follicles in the ovaries showed no perceptible differences between control and treated animals.

Dose descriptor:

NOAEL

Remarks:

adult toxicity

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: statistically significant reduction in body weight gain in F0 males, absolute and relative kidney weight increased in F0 and F1 males and absolute and relative liver weights in F1 males

Remarks on result:

other: Generation not specified (migrated information)

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: treatment had no effect on mating, fertility, gestation, fecundity, delivery or sperm parameters. No effects were observed on the progeny from delivery until weaning

Remarks on result:

other: Generation not specified (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Parental/adult observations:
F2 GENERATION:
One control female found dead on day 25, necropsy signs consistent with mis-dosing.
Ptyalism (excessive salivation) observed in all treated groups (dose-related trend), considered related to the unpalatable nature of the dosing solutions by study director.
Body weight and food consumption unaffected by treatment in both sexes.

LITTER SIZE AND PUP PARAMETERS DURING LACTATION
F0 LITTERS/F1 PUPS:
The number of litters delivered by the F0 dams and number of F1 pups per litter unaffected by treatment. At 250 mg/kg/d and 500 mg/kg/d there was a slight increase in the number of pups that died during the first 4 days of lactation, however findings for the high dose group were unremarkable and the findings were considered unrelated to treatment by the study director. Viability and lactation indices were unaffected by treatment at all dose levels.
0 250 500 1000 mg/kg/d
Litters obtained 23 21 22 25
Pups/dam 14.3 14.1 14.9 14.2
Viability index on LD4 (%) 97.6 92.9 82.3 97.7
Total decedent pups (LD1-21) 18 35 64 10
Lactation index (%) 94.6 91.7 96.1 99.0

F1 LITTERS/F2 PUPS
The number of litters delivered by the F1 dams and number of F2 pups delivered per litter unaffected by treatment. At 250 mg/kg/d and 1000 mg/kg/d, the number of pups that died during the lactation period was slightly higher than controls during the first 4 days of lactation. Considered unrelated to treatment by study director since only one (particularly large) litter per dose group affected. Viability and lactationindices unaffected by treatment at all dose levels.
0 250 500 1000 mg/kg/d
Litters obtained 21 21 22 20
Pups/dam 13.7 13.7 13.7 14.0
Viability index on LD4 (%) 97.6 94.8 97.0 92.9
Total decedent pups (LD1-21) 11 17 9 21
Lactation index (%) 97.6 98.8 100.0 99.3

PUP CLINICAL SIGNS, BODY WEIGHTS AND EXTERNAL FINDINGS
F1 PUPS
Pups from one low dose litter and three mid dose litters emaciated in appearance, cold to the touch or dehydrated, and were found dead or prematurely sacrificed for ethical reasons. Similar findings also observed in four surviving pups from three litters at 250 mg/kg/day, three surviving pups from one litter at 500 mg/kg/day and six pups from four different litters at 1000 mg/kg/day. Most of these pups displayed lower body weight. Since incidence was not dose related and did not correlate with lower mean group body weight gain, findings considered unrelated to treatment by the study director.
Body weight and body weight gain unaffected by treatment.
No gross external abnormalities present in F1 pups born to F0 mothers.

F2 PUPS
Tremors and ataxia present from day 15 post-partum in one pup from the 250 mg/kg/day group (sacrificed for humane reasons). Other clinical signs (necrosis of the tail or of the limbs,cold to the touch, emaciated appearance) were low in incidence, not dose-related and randomly distributed between the litters and treatment groups.
No effect on body weight or body weight gain noted at 250 and 500 mg/kg/day, while high dose pups exhibited a transient non-significant decrease (-11%) in body weight gain during the first 4 days after birth.
At 1000 mg/kg/day, absence of tail (acaudia) observed at birth in two female pups from two different litters, with anal atresia also present in one of these pups. The incidence of acaudia (2/280 pups; 0.7%) was outside the historic control range for the laboratory (minimum = 0.0%; maximum = 0.05%) data from 16 studies, October 2000-December 2003) but close to the background incidence recorded in the MARTA [*] historical control database (0.31% for 22147 fetuses from 1575 control litters during the period 1990-2004). The study director concluded that no clear relationship to treatment was established. No historical control data were available from the laboratory performing the study for anal atresia, however the observed incidence (1/280 pups; 0.35%) was close to that recorded in the MARTA historical control database (0.32% for 22147 fetuses from 1575 control litters during the period 1990-2004). Occurrence of anal atresia in this investigation considered spontaneous in origin by the study director.

[* A historical control database of preclinical developmental teratology and reproductive toxicity parameters, a joint project of MARTA and MTA; www.hcd.org]

Comment: No instance of acaudia or anal atresia in off-spring from dams treated with ETBE at a dose of 1000 mg/kg/day during a preliminary dose-range finding study (zero incidence in 164 fetuses from 12 litters and 144 pups from 11 litters; CIT study 24168 RSR, October 2003; summarised in this IUCLID dataset) or a developmental toxicity study (zero incidence in 258 fetuses from 22 litters; CIT study 24860 RSR, January 2004; summarised in this IUCLID dataset).

PUP ANOGENITAL DISTANCE, REFLEX DEVELOPMENT AND SEXUAL MILESTONES:
F1 PUPS:
Anogenital distance, the ratio of anogenital distance to pup body weight and anogenital distance normalized to the cube root of pup body weight not significantly altered by treatment.
Mean AGD values (in mm, SD in brackets) were:
0 250 500 1000
Males 4.71 (0.43) 4.62 (0.33) 4.47 (0.39) 4.64 (0.30)
Females 2.84 (0.36) 2.77 (0.24) 2.63 (0.26) 2.68 (0.37)

No evidence of any adverse treatment-related changes in surface righting, cliff avoidance and air righting (95 to 100% positive responses). (Observations performed on F1 pups only.)
Mean age at preputial separation unaffected by treatment (i.e. occurred on day 35, 34, 35 or 35 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively).
Mean age for vaginal opening unaffected by treatment (i.e. occurred on day 34, 34, 35 or 33 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively).
Auditory function (acoustic startle reflex) and visual function (pupil constriction reflex) at 4 weeks of age, and spontaneous locomotor activity (measured twice at 7 and 8 weeks old) unaffected by treatment. (Observations performed on F1 pups only)

F2 PUPS:
Anogenital distance, the ratio of anogenital distance to pup body weight and anogenital distance normalized to the cube root of pup body weight not significantly altered by treatment. Mean AGD values (in mm, SD in brackets) were:
0 250 500 1000
Males 4.57 (0.39) 4.56 (0.42) 4.54 (0.42) 4.63 (0.38)
Females 2.80 (0.35) 2.72 (0.29) 2.82 (0.29) 2.90 (0.33)

No evidence of any treatment-related effect on surface righting, cliff avoidance and air righting (between 96.0 and 99.4% positive responses).
Mean age at preputial separation similar in the control and the treated groups (i.e. occurred on day 36, 35, 35 or 36 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively).
Mean age for vaginal opening similar in the control and the treated groups (i.e. occurred on day 35, 34, 34 or 33 for pups from the control, 250, 500 and 1000 mg/kg/day groups, respectively). Although pups from two high dose dams displayed acaudia and anal atrasia, anogenital distance was normal.

NECROPSY AND HISTOPATHOLOGY:
F1 PUPS:
No treatment-related or statistically significant changes in pups found dead or sacrificed early, or in pups subject to scheduled necropsy at weaning.

F2 pups:
No treatment-related or statistically significant changes present in pups found dead or sacrificed early, or in pups subject to scheduled necropsy at weaning.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: statistically significant reduction in body weight gain in F0 males, absolute and relative kidney weight increased in F0 and F1 males and absolute and relative liver weights in F1 males

Reproductive effects observed:

not specified

In the two-generation study ptyalism was observed at all dose levels (i.e., from 250 mg/kg bw/day onwards). The study authors considered this effect related to unpalatable nature of the dosing solutions / to the unpalatable 'taste' of ETBE. In the EU RAR this effect is not taken into account as an adverse effect, therefore, this effect a not considered a significant health effect.

Conclusions:

The following No Observed Adverse Effect Levels were established from the study:
Systemic toxicity in the adult (parental) F0 and F1 generations: NOAEL = 250 mg/kg body weight/day (based on body weight and organ weight changes at higher treatment levels).

Ptyalism (excess salivation) was noted in all treated animals (LOEL = 250 mg/kg body weight/day but was not considered to represent an adverse effect of treatment.

Fertility, gonadal function, reproductive performance, parturition and lactation in the parental generations, and development of the off-spring to weaning or sexual maturity: NOAEL = 1000 mg/kg body weight/day (the highest dose tested).

Executive summary:

The test item, ETHYL TERTIARY BUTYL ETHER (ETBE), CAS No. 637-92-3, was
administered daily by oral gavage to male and female Sprague-Dawley rats at 250, 500 and 1000 mg/kg/day, commencing 10 weeks prior to mating and continuing through mating and gestation until the end of lactation in both the F0 and F1 generations. Progeny of the F1 generation (F2 pups) were treated from weaning until sexual maturity.

For all generations, ptyalism (excessive salivation) was observed with a dose-related trend in both males and females. At 1000 mg/kg/day, F0 males showed significantly lower body weight gain at the end of the dosing period. Liver weight was significantly increased in males only, with slight to moderate centrilobular hepatocellular hypertrophy in tissue from animals subject to microscopic
examination. Kidney weights were also significantly increased in F0 parental males, with acidophilic globules detected after microscopic examination. There were no adverse findings for F0 pups. Significantly greater food consumption during the lactation period was the only finding of note in F0 parental females.

Liver and kidney weights were significantly increased in F1 parental males.
Body weight gain of pups born to mothers from the F1 generation was slightly but
non-significantly lower than the controls on post-partum days 1-4 (no comparable finding in F0 litters). Two pups born to mothers from the F1 generation exhibited gross external malformations (absence of tail with anal atresia also present in one pup), however the incidence of these findings was comparable to laboratory or external historical control data. Neither malformation was present in 566 pups or fetuses from 45 litters from dams treated with ETBE at 1000 mg/kg body weight/day as part of a dose-range finding study and a developmental toxicity
study performed at this laboratory. It was concluded that the findings from the present study were therefore most probably unrelated to treatment with the test item.

No effects were noted in the F2 generation at 1000 mg/kg/day. At 500 mg/kg/day, significantly lower body weight gain was noted at the end of the dosing period in F0 parental males together with significantly increased kidney weights. Liver and
kidney weights were statistically significantly increased in F1 parental males, whereas body weight was unaffected. No effects were noted in the F2 generation.
At 250 mg/kg/day, no relevant findings were observed in the F0, F1 and F2 generations.

Based on these observations, the following No Observed Adverse Effect Levels were established from the study:
Systemic toxicity in the adult (parental) F0 and F1 generations: NOAEL = 250 mg/kg body weight/day (based on body weight and organ weight changes at higher treatment levels).

Ptyalism (excess salivation) was noted in all treated animals (LOEL = 250 mg/kg body weight/day but was not considered to represent an adverse effect of treatment.

Fertility, gonadal function, reproductive performance, parturition and lactation in the parental generations, and development of the off-spring to weaning or sexual maturity: NOAEL = 1000 mg/kg body weight/day (the highest dose tested).

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Information and data supporting the read-across hypothesis can be found in the supporting attachment in the 'attached justification section'. This includes the hypothesis for the analogue approach, source and target chemicals (including purity information), analogue approach justification and a data matrix.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Dose descriptor:

NOAEL

Remarks:

adult toxicity

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: statistically significant reduction in body weight gain in F0 males, absolute and relative kidney weight increased in F0 and F1 males and absolute and relative liver weights in F1 males

Remarks on result:

other: Generation not specified (migrated information)

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: treatment had no effect on mating, fertility, gestation, fecundity, delivery or sperm parameters. No effects were observed on the progeny from delivery until weaning

Remarks on result:

other: Generation not specified (migrated information)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: statistically significant reduction in body weight gain in F0 males, absolute and relative kidney weight increased in F0 and F1 males and absolute and relative liver weights in F1 males

Reproductive effects observed:

not specified

Conclusions:

The following No Observed Adverse Effect Levels were established from the study:
Systemic toxicity in the adult (parental) F0 and F1 generations: NOAEL = 250 mg/kg body weight/day (based on body weight and organ weight changes at higher treatment levels).

Ptyalism (excess salivation) was noted in all treated animals (LOEL = 250 mg/kg body weight/day but was not considered to represent an adverse effect of treatment.

Fertility, gonadal function, reproductive performance, parturition and lactation in the parental generations, and development of the off-spring to weaning or sexual maturity: NOAEL = 1000 mg/kg body weight/day (the highest dose tested).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable study available for the read-across source substance ETBE as well as a supporting OECD 422 study for TBHP itself. Key NOAEL provided is that for the multi-generational study on ETBE as is deemed to be the most robust study in assessing the fertility endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The test substance used in investigations supporting this endpoint where applicable was TBHP (aqueous 70% solution): all results have been expressed in terms of dry TBHP.

In an oral screening study (TNO Study Director, 1993) no toxicological relevant effects on fertility, reproductive performance or development were found at 21 mg TBHP/kg bwt/day, the highest dose tested. The use of higher treatment levels was not feasible given the occurrence of gastric damage in dose range finding studies that accompanied this investigation. Toxicokinetic data indicate rapid conversion of TBHP to tert-butanol in vivo limiting direct exposure of reproductive tissue to TBHP. Toxicokinetic data demonstrate very low or absent systemic bioavailability.

A multi-generational study on the read-across source substance ETBE is included in the dossier to meet the EOGRTs endpoint requirement for TBHP. Full details and justifications on the read-across are included in the appropriate target substance record in section 7.8.1 of IUCLID.

Effects on developmental toxicity

Description of key information

An oral PNDT study is available on TBHP conducted in rats. The NOAEL for the study was detailed as 35 mg/kg/day which was the highest dose tested. No effects were noted on developmental endpoints. The requirement for a non-rodent PNDT study is met using rabbit data available for the read-across source substance ETBE.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

ETBE was administered by oral gavage to pregnant female New Zealand White rabbits (Kbl:NZW) at dose levels of 0, 100, 300 and 1000 mg/kg bw/d on GD 6- 27, and effects on embryo-foetal development assessed.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Kbl:NZW
- Source: Kitayama Labs Co Ltd, Japan
- Age at study initiation: 17-18 wk
- Weight at study initiation: mean group weights approx. 3.25 kg
- Housing: single housed in aluminium cages
- Diet (ad libitum): RC4 pellets (Oriental Yeast Co. Ltd, Chiba, Japan)
- Water (ad libitum): domestic tap water
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 degrees
- Humidity (%): 50-72%
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 June 2007 (initiation of treatment) To: 3 July 2007 (start of cesarean section)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions prepared at 60, 180 and 600 mg/l in olive oil.
- Frequency of preparation: at least weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: test solutions were analyzed using GC-FID (Agilent Technologies HP6890N). Results were within 5% of target.

Details on mating procedure:

Females judged to be in oestrus (based on appearance of external genitalia) were housed together with untreated males on a 1:1 basis. Animals were considered pregnant when copulation was observed twice (designated GD 0).

Duration of treatment / exposure:

GD 6-27 (22 days)

Frequency of treatment:

daily, 7 d/wk

Duration of test:

GD 6-28

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: treatment levels were based on a preliminary study in which ETBE was administered orally to pregnant rabbits (6 / group) at 0, 30, 100, 300 and 1000 mg/kg bw/d for 22 days. Reduced food intake was noted at 1000 mg/kg bw/d but embryo-foetal development was unaffected. No maternal or foetal effects recorded at 300 mg/kg bw/d or below. Therefore the highest dose level was set at 1000 mg/kg bw/d with lower doses of 300 and 100 mg/kg bw/d.
- Rationale for animal assignment (if not random): stratified according to body weight
- Dosing: the control and test solutions were administered at 1.67 ml/kg body weight

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Dams were observed for clinical signs dosing, immediately after dosing and 1 hour post-dosing during the treatment period.

BODY WEIGHT:
Dams were weighed on GD 0 (at time of mating) and on GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. Any dams sacrified early were weighed before necropsy.

FOOD CONSUMPTION:
Food intake was measured on GD 1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.

NECROPSY:
Animals were sacrificed by exsanguination (abdominal aorta) under pentobarbital sodium anesthesia, and the major organs in the thoracic and abdominal cavities examined macroscopically. Animals were examined to confirm pregnancy. No organ weights measurement or routine tissue sampling were performed.

Ovaries and uterine content:

The ovaries and uterus were removed from dams confirmed as pregnant, and the number of corpora lutea determined. The uterus was weighed, opened, and the number of live/dead foetuses, implantation sites, resorbed embryos, placental remnants and early/late macerated foetuses counted. The placenta was also examined. Uteri were immersed in 10 vol% ammonium sulphide solution if no signs of pregnancy were visible macroscopically.

Fetal examinations:

- Body Weight:
Recorded for all foetuses (including those with external malformations, but the data excluded from group calculations).

- Sex Determination:
Assessed from examination of the internal genital organs of all live foetuses (excluding any with external malformations).

- External examinations
Foetuses were examined for the presence or absence of external malformations, including those in the oral cavity. Any exhibiting external malformations were preserved (phosphate buffered 10 vol% formalin).

- Soft tissue examinations:
The contents of the thoracic and abdominal cavities were observed macroscopically for all live foetuses (excluding those with external malformations). The brain (Wilson technique) and heart (Nishimura microdissection technique) were excised and fixed (phosphate buffered 10% formalin).

- Skeletal examinations:
All live foetuses (excluding those with external malformations) were fixed (95% alcohol), stained with Alizarin red S (Dawson method), and examined for skeletal malformations or variations. The degree of ossification was determined by counting the number of ossified metacarpals, metatarsals and digital phalanges and sacral/caudal vertebrae together with the degree of ossification of the sternebrae.

Statistics:

Body weight, body weight gain, food consumption, uterine weight, number of corpora lutea, number of implantations, number of live foetuses, sex ratio, body weight and ossification data (phalanges and vertebrae) were analyzed by Bartletts test followed by Dunnetts test if the data were homogeneous, with a Dunnett-type mean rank test used if the results were heterogeneous. Implantation indices, embryo-fetal deaths, external malformations, visceral malformations, visceral variations, skeletal malformations and skeletal variations were calculated and the data were analyzed by Dunnett-type mean rank test.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
GENERAL:
There were 1 or 2 were non-pregnant animals in the control, 100 and 300 mg/kg bw/d groups (all animals pregnant at 1000 mg/kg bw/d). No deaths occurred in any group. Abortions occurred in the control (1 animal on GD 28), 300 mg/kg bw/d (2 animals on GD 19 and GD 26) and 1000 mg/kg bw/d groups (1 animal on GD 25), but no dose-dependence apparent. One control animal and one animal that aborted in the 300 mg/kg bw/d group exhibited reductions in body weight and food intake for at least 4 days before abortion whereas clinical signs, body weight and food consumption were unremarkable in the other animals.

CLINICAL SIGNS:
Apart from reduced production of faeces (recorded in 14, 10, 7 and 13 animals from the 0, 100, 300 and 1000 mg/kg bw/d groups) there were no findings of note.

BODY WEIGHT:
Body weight was statistically significantly decreased by 4-5% in the 1000 mg/kg bw/d group relative to controls on GD 12, 14 and 16 but was unremarkable thereafter; body weight gain in this group on GD 6-28 was approx. half that of the control, however the effect was not statistically significant. Body weight and weight gain in the 100 and 300 mg/kg bw/d groups was unremarkable.

FOOD CONSUMPTION:
Food consumption was decreased significantly by 25-30% at 1000 mg/kg bw/d on GD 8, 10, 12 and 14, but was comparable to the control group from GD 20 onwards. Food intake for the 100 and 300 mg/kg bw/d groups was similar to that of the controls.

NECROPSY:
Dark red discoloration of the lung was observed in 1 animal from the 300 and 1000 mg/kg bw/d groups with mild to moderate haemorrhage and inflammatory cell infiltration noted microscopically; cause not known but judged of limited limited relevance. There were no other findings of note.

CESAREAN SECTION DATA:
The number of corpora lutea, implantations and implantation index was similar in the control and treated groups. Uterus weight was comparable between the groups.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
GENERAL:
There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

VISCERAL EXAMINATION:
No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

SKELETAL EXAMINATION:
No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

  Selected results:

Treatment (mg/kg bw/d)	0	100	300	1000
Mean no. corpora lutea	10.3	10.4	10.1	9.8
Mean no. implantations	8.4	8.7	9.0	7.7
Mean implantation index (%)	80.4	84.7	89.3	77.1
Mean no. resorbed foetuses	11.0	11.3	7.0	8.7
Total no. early resorptions	8	7	10	11
Total no. late resorptions	5	10	2	8
Mean no. live foetuses	7.8	7.9	8.4	6.9
Sex ratio	0.50	0.54	0.54	0.42
Male foetal bw (g)	33.5	33.4	33.9	32.3
Female foetal bw (g)	31.2	31.4	32.0	30.1
No. foetuses examined	171	173	167	158
No. visceral malformations	1	1	1	3
No. visceral variations	3	5	5	3
No. skeletal malformations	5	4	3	8
No. skeletal variations	9	11	6	15

 

Conclusions:

Dams in the 1000 mg/kg bw/d group showed small but statistically significant reductions in body weight on GD 8-14 (the period of organogenesis) with significant reductions in food intake over the same period; overall body weight gain on GD 6-28 was decreased by one half. The maternal NOAEL was therefore 300 mg/kg bw/d. There were no effects of ETBE-treatment on pregnancy or foetal development (NOAEL = 1000 mg/kg bw/d).

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Information and data supporting the read-across hypothesis can be found in the supporting attachment in the 'attached justification section'. This includes the hypothesis for the analogue approach, source and target chemicals (including purity information), analogue approach justification and a data matrix.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

 

 

Conclusions:

Dams in the 1000 mg/kg bw/d group showed small but statistically significant reductions in body weight on GD 8-14 (the period of organogenesis) with significant reductions in food intake over the same period; overall body weight gain on GD 6-28 was decreased by one half. The maternal NOAEL was therefore 300 mg/kg bw/d. There were no effects of ETBE-treatment on pregnancy or foetal development (NOAEL = 1000 mg/kg bw/d).

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, acceptable for assessment.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Pregnant female rats were administered TBHP by oral gavage on gestation days 6-15, sacrificed on GD21 and the fetuses assessed for visceral- and skeletal abnormalities.

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatstoezicht op de Volkgezondheid, 19 April 1989; 6 June 1991

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wigd GmbH, Sulzfeld, Germany
- Age at study initiation: approx. 12 wk
- Weight at study initiation (females, GD0): 178.8-239.1 g
- Housing: individually (during acclimation and pregnancy) or in pairs of one male and one female (during mating)
- Diet (e.g. ad libitum): cereal-based stock diet ad libitum
- Water (e.g. ad libitum): tap-water ad libitum
- Acclimatisation period: 7 d

Males (age 12 wk) of the same strain and from the same supplier were used for mating

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Solutions of test substance were prepared in Millipore-filtered water and stored in brown glass bottles, in a refrigerator, until use.

Concentration in dosing solutions (mg Aq. TBHP-70/ml): 0, 0.5, 1.5, 5
Equivalent as TBHP, mg/ml: 0, 0.35, 1.05, 3.5

Dosing volume: 10 ml/kg bwt

The test substance was administered once daily by oral gavage (10 ml/kg) with the dose volume were adjusted for changes in body weight. Two batches of dosing solutions were prepared (on June 29 and July 13 1992).

The in-life phase commenced on 22 June 1992 and was terminated on 29 July 1992.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were analysed by GC-FID using a Perkin Elmer 8500 gas chromatograph with fused silica 50 m x 0.21 mm column and 0.5 um film methylsilicon PONA. Stability was determined at room temperature for 3 days or in a refrigerator for up to 16 days. Homogeneity was checked by analysing samples from the top, middle and bottom of the dosing solutions. Results demonstrated that achieved concentrations were nominal.

Details on mating procedure:

One female was placed with one male from the same treatment overnight. A vaginal smear was taken the following morning and the presence of sperm or a vaginal plug was used to designate GD0. Females were returned to their home cage after mating.

Duration of treatment / exposure:

Pregnant females were treated on GD5-16, and sacrificed on GD21.

Frequency of treatment:

Daily

Duration of test:

Pregnant females were treated on GD5-16, and sacrificed on GD21.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection was based on range-finder tests described in Chapter 7.5.1, record Repeated dose toxicity: oral.001 (TNO report V 92.494)

The test substance was administered to pregnant female rats once daily on GD5-16 followed by necropsy on GD21.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once (weekends) or twice (week days) per day

BODY WEIGHT: Yes
- Time schedule for examinations: GD0, 6, 11, 16, 21

FOOD CONSUMPTION: Yes
- Food consumption determined: GD 0-6, 6-11, 11-16, 16-21

WATER CONSUMPTION : not recorded

POST-MORTEM EXAMINATIONS: Yes
- sacrifice on gestation day 21
- microscopic examination of major abdominal and thoracic organs

Ovaries and uterine content:

- Examinations performed:
- number of corpora lutea, implantation sites, early/late resorptions
- weight of ovaries, uterus, placenta

Fetal examinations:

- Examinations performed:
- number of live, dead and grossly malformed fetuses
- fetal weights
- length and sex of fetuses

Approx. on half of each litters was fixed (Bouins) and subsequently examined for visceral alterations (Wilson technique). The remainder was fixed (70% ethanol) and examined for skeletal changes (Alizarin red staining).
Note: examinations limited to control and high dose groups only since no treatment-related changes present

Statistics:

Body weights, food consumption, fetal body weights/lengths and placental weights were subject to one way ANOVA followed by Dunnett’s multiple comparison test. Clinical findings, mating data were evaluated by Fisher’s exact probability test. The numbers of corpora lutea, implantation sites, resorptions and fetuses were analysed by the Kruskal-Wallis test followed by the Mann Whitney U-test.

Indices:

Early resorptions = only placental tissue visible
Late resorptions = placental and embryonic tissue visible
Pre-implantation loss = (corpora lutea - total implantation sites)/corpora lutea x 100%
Post-implantation loss = (total implantation sites - live fetuses)/total implantation sites x 100%

Historical control data:

Not used

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
None present

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
None present

Dose descriptor:

NOAEL

Effect level:

>= 35 mg/kg bw/day (actual dose received)

Basis for effect level:

other: No effect on number of litters, live foetuses, live litter size, sex ratio, body weight, crown-rump length, visceral effects (malformation, anomalies, variations) or skeletal changes (malformations, anomalies, variations, ossification delays).

Abnormalities:

not specified

Developmental effects observed:

not specified

MATERNAL DATA

All dams survived to scheduled necropsy on GD21 with no treatment-related clinical signs of effects on food consumption.

Net maternal body weight (corrected for gravid uterus weight )was similar in all groups, while net body weight gain was slightly but non-significantly reduced in the intermediate and high dose groups (values shown below as mean and (SD), by dose level):

Net maternal body (g): 253.00(3.00), 255.02(3.33), 246.69 (4.64), 252.24(3.67)

Net body weight gain (g): 40.37(2.08), 40.44(2.11), 33.87(2.26), 35.58(2.23)

No unusual gross maternal findings were detected at necropsy.

REPRODUCTION DATA

Female fertility (in a range 83% (5 mg/kg bwt/d) to 92% (all other groups)) was unaffected by treatment.

There were no abortions, and all females had litters containing viable foetuses. There was no statistically significant difference in mean gravid uterine weight, empty uterine weight or mean ovary weight.

The mean numbers of corpora lutea, implantation sites and resorptions did not differ between the groups.

LITTER DATA

The number of litters, total live foetuses, live litter size, sex ratio (%), fetal body weight (g) and fetal crown-rump length (cm) were not affected by treatment (results presented below, by dose level):

Number of litters: 21/24, 19/24, 21/24, 21/24

Total live foetuses: 220, 201, 232, 220

Total dead foetuses: 0, 0, 0, 0

Live litter size (SD): 10.48(0.39), 10.63(0.40), 11.05(0.49), 10.48(0.41)

Males: 50, 52, 50, 53

Body weight (SD): 4.79(0.06), 4.76(0.07), 4.72(0.06), 4.81(0.08)

Crown-rump length (SD): 4.08(0.02), 4.09(0.02), 4.11(0.03), 4.10(0.03)

VISCERAL AND SKELETAL EXAMINATIONS

Examination of foetuses from the control and high dose group revealed no treatment-related change in the number of visceral malformation, anomalies and variations, with any changes present either found in both the control and treated groups or of a type considered by the study director to be within normal limits.

Total visceral malformations:

- litters: 0/21, 0/21

- fetuses: 0/104, 0/105

Total visceral anomalies:

- litters: 3/21, 5/21

- fetuses: 5/104, 6/105

Total visceral variations:

- litters: 9/21, 13/21

- fetuses: 11/104, 19/105

Similarly there was no treatment-related effect on the number of skeletal malformations, anomalies or variations in ossification in the control and high dose groups, with any changes present either found in both the control and treated groups or of a type considered by the study director to be within normal limits.

Total skeletal malformation:

- litters: 0/21, 0/21

- fetuses: 0/104, 0/105

Total skeletal anomalies:

- litters: 17/21, 18/21

- fetuses: 39/104, 36/105

Total skeletal variations:

- litters: 1/21, 1/21

- fetuses: 2/104, 1/105

Total skeletal retardation of ossification:

- litters: 21/21, 21/21

- fetuses: 116/104*, 115/105*

* = more than one finding per fetus

Conclusions:

TBHP is not selectively toxic to the fetus.

Executive summary:

The developmental toxicity of TBHP was investigated in a GLP-compliant guideline study (OECD 414)  in which groups of pregnant female Wistar rats (24/dose level) received TBHP by oral gavage in water at nominal doses of 0, 5, 15 or 50 mg/kg bw/day on GD5 -16 (actual doses = 0, 3.5, 10.5 or 35 mg TBHP/kg bwt/day). The dams were sacrificed on GD21, and a full range of maternal, reproductive and fetal parameters evaluated. Dose levels used in this study were based on results obtained from two preliminary range-finding studies which demonstrated moderate to severe damage to the stomach lining at higher dose levels. Overall there were no statistically significant or biologically relevant findings leading to maternal and fetal NOAELs of 35 mg TBHP /kg bwt/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

35 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable PNDT study available on TBHP and in rabbits for the read-across source substance ETBE. Key NOAEL is provided for the rat PNDT study as was conducted on TBHP itself using the same guideline as the rabbit study on ETBE.

Full details and justifications on the read-across are included in the appropriate target substance record in section 7.8.1 of IUCLID.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No adverse effects on development or litter parameters were found following oral exposure of pregnant rats to TBHP at doses up to 35 mg/kg bwt/day (TNO Study Director, 1993). The use of higher treatment levels was not feasible given the occurrence of gastric damage at higher oral doses while toxicokinetic data indicate rapid conversion of TBHP to tert-butanol in vivo limiting direct exposure of the foetus to TBHP.

The test substance used in this investigation was TBHP (aqueous 70% solution): results have been expressed in terms of dry TBHP.

No adverse effects on development or litter parameters were observed in the available rabbit PNDT study in the source substance ETBE with the NOAEL set as the highest dose tested (1000 mg/kg/day). Full details and justifications on the read-across are included in the appropriate target substance record in section 7.8.1 of IUCLID.

Justification for classification or non-classification

Toxicokinetic data demonstrate rapid conversion of TBHP to TBA in vivo limiting the potential for exposure of gonadal tissue. Results from a rat oral reproduction screening study and a rat oral developmental toxicity test in TBHP demonstrate no adverse effects on fertility, reproductive performance or foetal development. Read-across has been used on the source substance ETBE to fulfil the information requirements on TBHP in relation to a multi-generational study and non-rodent PNDT study. Both studies on ETBE showed no adverse effects in reproductive or developmental parameters. Full details and justifications for the read-across can be found in the relevant endpoint sections.

Toxicokinetic data indicate that TBHP is efficiently absorbed and excreted based on comparable kinetic parameters for total radioactivity after oral gavage, iv and sc exposures together with the high urinary excretion of TBHP-related radioactivity. However, the bioavailability of TBHP itself (ie presence of parent substance in the systemic circulation) is very low or absent due to its rapid conversion to 2-methylpropan-2-ol, as demonstrated by the absence of TBHP in the blood shortly after iv injection. This absence of systemic bioavailability is consistent with the available toxicological data which indicate only local toxicity and no systemic effects.

In an OECD 422 investigation (combined repeat dose toxicity with reproduction / developmental screening), no toxicologically relevant effects on fertility, reproductive performance or development were found following gavage administration of TBHP at doses of 2.1, 7 or 21 mg/kg bwt/d. Treatment commenced 2 weeks prior to mating and continued through pregnancy for up to 45 consecutive days.

Results from an OECD 414 prenatal developmental toxicity study showed no adverse effects on foetal development or litter parameters following oral exposure of pregnant rats to TBHP at doses of 3.5, 10.5 or 35 mg/kg bwt/day. Treatment commenced on GD5 and continued to GD16, with sacrifice of the dams and examination of the uterine contents occurring on GD21. Foetuses were assessed for the presence of external malformations together with a detailed inspection for the presence of visceral or skeletal malformations, anomalies and variations.
No evidence of systemic toxicity was recorded when TBHP was administered by oral gavage to rats at doses of up to 21 mg/kg bwt/d for up to 45 days, or in rats exposed via inhalation to 66.7 mg/m3 (6 hr/d, 5 d/wk for 4 wk). Local toxicity, demonstrated by damage to the lining of the stomach or the transitional epithelia of the nasal cavity, respectively, was apparent in both studies. The occurrence of local toxicity precluded the use of higher treatment levels in the reproduction and developmental toxicity studies described above.

No adverse effects were noted in the available multi-generational study on ETBE with the NOAEL set as the limit dose (1000 mg/kg/day). No adverse effects were also observed in the available rabbit PNDT study on ETBE together indicating no effects on reproductive or developmental parameters are expected for the target substance TBHP. Full details and justifications for the read-across can be found in the relevant endpoint sections.  

No classification is deemed to be required under the CLP Regulation (EC No.1272/2008, as adapted).