Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Only repeated toxicity studies are available.

Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Elements of screening for the reproductive toxicity potential of 4-tert butylpyrocatechol (TBC) were obtained from the 3-month dietary toxicity studies in both rats and mice, where sperm and vaginal cytology observations were performed (ref. 1).

 

A reproductive toxicity alert, consisting of altered sperm and estrous cycle parameters, was observed. Significant reductions in male reproductive organ weights and sperm count or motility were noted in male rats at the highest dose level of 12500 ppm after 14 weeks of administration. In female rats, significantly fewer estrous cycles than controls were seen at all dose levels assessed (from 3125 ppm) and the estrous cycle duration increased with concentration, the females spending more time in diestrus than controls. In female mice also, significantly longer estrous cycles were seen at the highest dose level of 12500 ppm.

 

Some of these reproductive changes may be the result of a systemic toxicity, such as effects on body weight and food consumption, based on the results of feed restriction studies in the same rodent species (ref. 2 & 3). However systemic toxicity on its own can not account for all the changes seen in reproductive parameters and their magnitude. So a direct effect of TBC on sperm and estrous cycle parameters, with a potential subsequent impact on fertility and pregnancy, can not be ruled out.

 

Furthermore, studies on close structural analogues of TBC, butylated hydroxyanisole and butylated hydroxytoluene, have shown that these substances are able to modify the effects of chemically induced mammary gland tumors. In the case of butylated hydroxyanisole, this was attributed, at least in part, to increasing the activities of liver microsomal enzymes catalyzing glucuronidation and oxydation of estradiol and estrone (ref. 4). It could be hypothesized that TBC may similarly affect estrogen levels in female rodents, interfering with estrous cycle duration. In vitro, TBC has been shown to exert a weak estrogenic activity (1/300 000 compared to 17β-estradiol) in a recombinant yeast receptor binding assay (ref. 5). The endocrine disruption potential of butylated hydroxyanisole has also been studied in rats treated at 10,100 or 500 mg/kg through gestation, lactation and development of offspring (ref. 6). Decreases in reproductive organ weights (vagina, testes and ventral prostate), delayed offspring sexual maturation, reduced steroid (testosterone) or thyroid (thyroxine) hormon levels, and thyroid histopathological changes (follicular cell height increase or vacuolation) were observed, mainly at 500 mg/kg.

 

At this stage of the evaluation, no sufficiently clear elements warrant any classification of TBC for reproductive toxicity. However, based on the reproductive alerts seen in the subchronic toxicity studies on TBC, and other elements of reproductive toxicity on its structural analogues, the performance of a extended one generation study (OECD TG 443) is recommended for further evaluation.

1. Dunnick J.K. NTP Technical Report on the Toxicity Studies of 'p-tert-Butylcatechol' (CAS No. 98-29-3) Administered in Feed to F344/N Rats and B6C3F1 Mice. Toxicity Report Series Number 70 (2002).

2. Chapin R.E., Gulati D.K., Barnes L.H. and Teague J.L. The effects of feed restriction on reproductive function in Sprague-Dawley rats. Fundam. Appl. Toxicol. 20: 23 -29 (1993).

3. Chapin R.E., Gulati D.K., Fail P.A., Hope E., Russell S.R., Heindel J.J., George J.D., Grizzle T.B. and Teague J.L. The effects of feed restriction on reproductive function in Swiss CD-1 mice. Fundam. Appl. Toxicol. 20: 15 -22 (1993).

4. Zhu B.T., Lech J., Rosen R.T., and Conney A.H. Effect of dietary 2(3)-tert-butyl-4-hydroxyanisole on the metabolism and action of estradiol and estrone in female CD-1 mice. Cancer Res. 57: 2419 -27 (1997).

5. Miller D, Wheals BB, Beresford N, Sumpter JP. Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay.Environ Health Perspect. 109(2): 133-8 (2001).

6. Jeong SH, Kim BY, Kang HG, Ku HO, Cho JH. Effects of butylated hydroxyanisole on the development and functions of reproductive system in rats. Toxicology. 208(1): 49-62 (2005).


Short description of key information:
Reproductive toxicity alerts in 3-month repeated dose toxicity studies in rats and mice, triggering testing proposal for extended one-generation (OECD TG 443) study.

Justification for selection of Effect on fertility via oral route:
A OECD 443 test guideline has been proposed as a testing proposal.

Effects on developmental toxicity

Description of key information

Two OECD 414 test guideline studies have been performed, one on rats (2013) and the other one on rabbits (2017).

The OECD 414 test guideline study performed on rats (CIT, 2013 - reliability 1) showed at 300 mg/kg/day (highest tested dose) non-statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations in a context of maternal toxicity. No malformation was noted and the increasing in fetal variations was considered to be of minor toxicological significance.

On the OECD 414 test guideline study performed on rabbits (CharlesRiver, 2017 - reliability 1), time-mated New Zealand rabbits were treated from day 6 to day 28 post-coitum by daily gavage at dose levels of 30, 30 and 100 mg/kg/day. No adverse developmental toxicity related to the substance was observed at all dose groups.

Therefore based on these two developmental studies TBC does not induce teratogenic effect.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 May 2013 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 414 compliant
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate n°2012/96 dated 10 January 2013
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Number: 96 female rats were received at CiToxLAB France between 22 and 31 May 2013.
- Strain and sanitary status: Sprague-Dawley, Rj Han: SD (IOPS Han).
- Breeder: Janvier, le Genest-Saint-Isle, France.
- Age/Weight: at the beginning of the treatment period, the females were 10-11 weeks old and had a mean body weight of 250 g (range: 202 g to 309 g). The females were sexually mature and primigravid.
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm², 48 cm x 26.5 cm x 21 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was selected in order to not jeopardize gestation.
Each cage contained an object for the environmental enrichment of the animals (rat hut).
The cages were placed in numerical order on the racks.
- Food and water: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 4898480 and 6318584 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation: the animals were acclimated to the conditions of the study for a period of 5 days before the beginning of the treatment period (arrival of the females on day 1 post-coitum (p.c.)).
- Allocation to study: during the acclimation period, the animals were allocated to the groups, according to a stratification procedure based on body weight recorded on day 2 p.c., to ensure comparatively similar mean body weights of the groups.
- Identification: each animal was individually identified by an ear tattoo (unique CiToxLAB France identity number).

ENVIRONMENTAL CONDITIONS
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: about 12 cycles/hour of filtered, non-recycled air.

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
DOSE FORMULATION PREPARATION
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, mixed with the required quantity of vehicle and kept under continuous stirring for at least 30 minutes.
The test item dose formulations were prepared for up to 7 days (stability results up to 11 days, CiToxLAB France/Study No. 39960 AHS), stored at room temperature and protected from light.
Dose formulations were delivered at room temperature and protected from light (in brown flasks).

VEHICLE
The vehicle was corn oil, batch No. MKBH4894V.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 39959 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.
Details on mating procedure:
- Mating: the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).

Duration of treatment / exposure:
The dose formulations were administered daily from day 6 to day 20 p.c., inclusive.
Frequency of treatment:
Daily
Duration of test:
One month
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2 week dose-range finding study by oral route (gavage) in rats (CiToxLAB France/Study No. 39961 RSR) where a total of 20 time-mated female Sprague-Dawley rats were allocated to 4 groups and received the test item at 50, 150 or 400 mg/kg/day, or the vehicle (corn oil) once daily from day 6 until day 20 post-coitum (p.c.).
In this study, one pregnant dam given 400 mg/kg/day was prematurely sacrificed on day 12 p.c. for ethical reasons (emaciated appearance, hypoactivity, loud breathing, abdominal breathing, dyspnea, ptyalism, eyes half-closed and 8% of body weight loss between days 6 and 9). At 150 mg/kg/day, all females had ptyalism and one had loud breathing. At 400 mg/kg/day all females had ptyalism and loud breathing. Furthermore, when compared with controls, there were marked decreases in mean body weight ( 17% vs. controls on day 21 p.c.) and mean body changes (-38% for the period of days 6 to 21 p.c.) associated with reduced mean food consumption at the same dose level (up to -61% for the period of days 6 to 9 p.c.). At hysterectomy, there were no obvious effects on mean reproductive parameters but a significant decrease in mean fetal body weight at 400 mg/kg/day (-12.5% vs. controls). There were no findings at external examination of the fetuses.

Therefore, 400 mg/kg/day was considered to be a dose-level associated with excessive maternal toxicity and 300 mg/kg/day was selected as the high dose-level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 100 mg/kg/day).

Maternal examinations:
MORBIDITY AND MORTALITY:
Each animal was checked for mortality and morbidity at least once a day before and after the treatment period and at least twice a day during the treatment period, including weekends.

CLINICAL SIGNS:
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
The body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

FOOD CONSUMPTION:
The quantity of food consumed by each female was recorded for the following intervals:
- days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21 p.c.
- Macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- Other organs examined: liver, vagina, colon, jejunum and thymus
Any macroscopic lesions observed were sampled and kept preserved in 10% buffered formalin The corresponding tissues of five control animals were sampled and preserved.
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with discernible digits.

A gross evaluation of placentas was also undertaken.



Fetal examinations:
Fetal examination was conducted for all litters where the female had at least one live fetus.

BOBY WEIGHT OF FETUSES:
The body weight of each live fetus was recorded.

SEX oF FETUSES:
The sex of each fetus (excluding any autolyzed fetuses) was determined at the time of hysterectomy.
The sex of fetuses was determined by visual assessment of anogenital distance and was confirmed by examination of sexual organs at detailed dissection of the soft tissues or at evisceration. Autolyzed fetuses were not sexed.

EXTERNAL EXAMINATION:
Each fetus (excluding any autolyzed fetus) was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

SOFT TISSUE EXAMINATION:
As soon as possible after sacrifice, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed with Harrison's fluid for examination of the structures of the head. A fetus was submitted to skeletal examination instead of visceral examination due to its fetal external malformation.

SKELETAL EXAMINATION:
The remaining live fetuses per litter were eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
PREGNANCY STATUS: At termination on day 21 p.c., there were 24, 24, 24 and 19 females with live fetuses in the groups treated at 0, 30, 100 and 300 mg/kg bw/day, respectively.

MORTALITY: There were no unscheduled deaths in control, 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day, five females (all pregnant) were found dead:
- two females were found dead on day 16 or 14 p.c., respectively. Loud breathing and ptyalism were noted from days 9 to 15 p.c. or 8 to 13 p.c. before death, respectively. At necropsy, both females had a transverse vaginal septum with a colored content (brownish thick),
- one female was found dead on day 18 p.c.. Loud breathing, ptyalism and abdominal breathing were noted from day 7 p.c. until death. At necropsy, this female had a dilated colon,
- one female was found dead on day 8 p.c.. No clinical signs were noted before death and there were no findings at necropsy,
- one female was found dead on day 7 p.c.. Loud breathing, ptyalism and hypoactivity were noted on day 7 p.c. before death. At necropsy, this female had reddish colored focus in thymus and reddish color liquid in intestines (jejunum).

All these deaths were considered to be associated to the test item treatment due to the incidence of mortality which was limited to the high dose.

CLINICAL SIGNS: At 300 mg/kg bw/day, hypoactivity, loud breathing and ptyalism were noted in most females.
Ptyalism noted at 100 and 300 mg/kg bw/day was considered to be of minor toxicological significance.

BODY WEIGHT: When compared with controls, there were no effects on mean body and mean body weight change at 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day and from day 6 to day 9 p.c. there was a body weight loss (-16 g vs. +11 g in controls, p<0.001). Thereafter, mean body weight gain returned towards control values but mean body weight remained lower than controls. When compared with controls, all differences were statistically significant (up to -12.9%, p<0.001).

FOOD CONSUMPTION: When compared with controls, there were no effects on mean food consumption at 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day, there was a marked decrease in mean food consumption on initiation of the treatment period (-57.1% vs. controls on the period of days 6-9 p.c.). Thereafter, mean food consumption remained lower than control (-17.2% vs. controls on days 18-21 p.c., p<0.001).

MACROSCOPIC post-mortem EXAMINATION: There were no test item treatment-related findings at macroscopic post-mortem examination.
In the 100 mg/kg bw/day group, one female had a liver with colored area. This finding was isolated and considered to be fortuitous.

NET BODY WEIGHT CHANGE: When compared with controls, there were no effects on mean gravid uterus weight, mean carcass weight and net body weight change at 30 and 100 mg/kg bw/day groups.
At 300 mg/kg bw/day and when compared with controls, females had lower mean gravid uterus weight (-13%), mean carcass weight (-13%) and mean net body weight gain from day 6 p.c. (-70%).

HYSTERECTOMY DATA (Table 1): When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg/day.
At 300 mg/kg bw/day and while not statistically significant, there were increased mean number of dead fetuses (higher than the upper limit of Historical Control Data) and a tendency towards dose-related increased mean post-implantation loss (within the range of Historical Control Data).
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHT AND SEX RATIO: When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg bw/day.
At 300 mg/kg bw/day, there was a decrease in mean fetal body weight, while treatment-related this slight finding (-7% vs. controls) was considered to be of minor toxicological significance.
There were no test item treatment-related effects on sex ratio.

EXTERNAL EXAMINATION (Tables 2 and 3): There were no external variations in the 30 and 100 mg/kg/day groups.
At 300 mg/kg bw/day and when compared with Historical Control Data, there was an increased litter incidence of fetuses with autolysis. However a similar finding was recorded in the contemporaneous control group and a test item treatment-related effect cannot be ascertained.
When compared with controls, there were no test item treatment-related findings in the number of fetuses with external malformations.

SOFT TISSUE EXAMINATION (Table 4): There were no malformations in control and test item-treated groups at soft tissue examination.

When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg bw/day.
At 300 mg/kg bw/day and when compared with controls, there was a statistically significant increase in the number of fetuses variations. This increase was considered to be associated to the treatment to the test item but of minor toxicological significance.

CARTILAGE AND SKELETAL EXAMINATION (Table 5): At cartilage examination and when compared with controls, there were a few statistical significant differences but none were of toxicological relevance.
There were no relevant malformations in control and test item-treated groups at skeletal examination.
When compared with controls, there were no test item treatment-related findings at 30 and 100 mg/kg bw/day. In the absence of any dose-relationship, the statistically significant increase in unossified hindpaws at 100 mg/kg bw/day was considered fortuitous.
At 300 mg/kg bw/day and when compared with controls or Historical Control Data, there was a statistically significant increase in the number of fetuses with ossification delays (thoracic vertebra(e) with dumbbell ossification centrum, incomplete ossification of the 1st to 4th sternebrae, metacarpals with incomplete ossification and/or unossified 1st metatarsal). This increase was considered to be associated to the treatment to the test item but of minor toxicological significance.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Table 1: Hysterectomy data

Dose-level (mg/kg bw/day)

0

30

100

300

HCD

Number of females with live fetuses
at termination

24

24

24

19

150

Mean number ofcorpora luteaper animal

15.3

14.8

14.8

14.7

14.0 - 15.5

Mean number of implantations per animal

14.0

13.7

13.4

13.6

12.8 - 14.0

Mean pre-implantation loss (%)

8.0

6.2

9.0

7.4

7.2 - 13.9

Mean number of fetuses per animal

13.8

13.3

12.8

12.8

12.0 - 13.2

Dead fetuses (%)

0.3

0

0

0.8

0.0 - 0.38

Mean number of implantation scars

0

0

0

0

0.3 - 1.0

Mean number of early resorptions

0.2

0.4

0.5

0.7

/

Mean number of late resorptions

0

0

0

0

/

Mean post-implantation loss (%)

1.5

3.0

3.8

6.2

2.0 - 8.7

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012).

/: not available in HCD.

Table 2: Litter (L) and Fetal (F) incidences of external variations

Dose-level (mg/kg bw/day)

0

30

100

300

HCD

Dams with live fetuses, n

24

24

24

19

143

Fetuses examined, n

332

319

308

244

1824

- Autolysis,L(F) %

4.2 (0.3)

0 (0.0)

0 (0.0)

10.5 (0.8)

8.3 (2.8)

Litters affected, n (%)

1 (4.2)

0 (0.0)

0 (0.0)

2 (10.5)

3 (2.1)

Fetus affected, n (%)

1 (0.3)

0 (0.0)

0 (0.0)

2 (0.8)

16 (0.9)

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (143 out of 150 litters had their fetuses submitted to external examination)).

Table 3: Litter (L) and Fetal (F) incidences of external malformations

Dose-level (mg/kg bw/day)

0

30

100

300

HCD

Dams with live fetuses, n

24

24

24

19

143

Fetuses examined, n

332

319

308

244

1824

- Agnathia,L(F) %

0 (0.0)

4.2 (0.3)

0 (0.0)

0 (0.0)

-

Litters affected, n (%)

0 (0.0)

1 (4.2)

0 (0.0)

0 (0.0)

0 (0.0)

Fetus affected, n (%)

0 (0.0)

1 (0.3)

0 (0.0)

0 (0.0)

0 (0.0)

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (143 out of 150 litters had their fetuses submitted to external examination)).

-: not present in HCD.

Table 4: Litter (L) and Fetal (F) incidences of soft tissues variations

Dose-level (mg/kg bw/day)

0

30

100

300

HCD

Dams with live fetuses, n

24

24

24

19

140

Live fetuses, n

159

153

149

115

861

- Cranial cavity, hematoma, L(F) %

0 (0.0)

4.2 (0.7)

4.2 (0.7)

5.3 (0.9)

-

- Liver, colored nodule, L(F) %

0 (0.0)

4.2 (0.7)

0 (0.0)

0 (0.0)

-

- Short innominate artery, L(F) %

0 (0.0)

4.2 (1.3)

4.2 (0.7)

5.3 (0.9)

8.3 (2.0)

- Absent innominate artery, L(F) %

0 (0.0)

0 (0.0)

0 (0.0)

5.3 (2.6)

-

Litters affected, n (%)

0 (0.0)

3 (12.5)

2 (8.3)

3 (15.8)

17 (12.1)

Fetus affected, n (%)

0 (0.0)

4 (2.6)

2 (1.3)

5* (4.3)

24 (2.8)

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (140 out of 150 litters had their fetuses submitted to soft tissues examination)).

-: not present in HCD.

*: p<0.05.

Table 5: Litter (L) and Fetal (F) incidences of skeletal variations

Dose-level (mg/kg bw/day)

0

30

100

300

HCD

Dams with live fetuses, n

24

24

24

19

141

Fetuses examined, n

172

165

159

127

930

- Interparietal, incomplete ossification, L(F) %

4.2 (0.6)

20.8 (3.0)

12.5 (3.1)

31.6* (5.5*)

40.0 (12.9)

- Thoracic vertebra(e),
 dumbbell ossification centrum, L(F) %

4.2 (0.6)

16.7 (2.4)

8.3 (1.3)

31.6* (6.3**)

21.7 (4.8)

- Caudal vertebra(e), unossified arch, L(F) %

4.2 (0.6)

0 (0.0)

0 (0.0)

10.5 (4.7*)

8.3 (2.5)

- Sternebra(e), incomplete ossification
 of the 1st to 4th, L(F)%

4.2 (0.6)

16.7 (3.0)

8.3 (1.3)

15.8 (7.1**)

10 (2.4)

- Sternebra, incomplete ossification of the 6th, L(F) %

4.2 (0.6)

4.2 (0.6)

0 (0.0)

21.1 (5.5*)

25.0 (7.4)

- Metacarpal(s), incomplete ossification, L(F) %

0 (0.0)

4.2 (1.2)

0 (0.0)

10.5 (7.1#)

12.5 (4.3)

- Metatarsal(s), 1st unossified, L(F) %

33.3 (9.3)

45.8 (11.5)

29.2 (6.9)

57.9 (21.3**)

37.5 (12.3)

- Hindpaw, unossified distal phalanx, L(F) %

29.2 (7.6)

41.7 (12.7)

54.2 (18.2**)

31.6 (7.9)

37.5 (20.2)

Litters affected, n (%)

24 (100.0)

24 (100.0)

23 (95.8)

19 (100.0)

122 (86.5)

Fetus affected, n (%)

113 (65.7)

102 (61.8)

95 (59.7)

80 (63.0)

377 (40.5)

Statistical significance: *: p<0.05, **: p<0.01, #: p<0.001.

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from February 2008 to March 2012 (141 out of 150 litters had their fetuses submitted to skeletal examination)).

Conclusions:
The test item, 4-Tert Butylpyrocatechol (batch No. C 492 L 2262), was administered by gavage, once daily, from days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosage of 30, 100 or 300 mg/kg bw/day.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on clinical signs, deaths, decreased body weight and decreased food consumption, and decreased mean carcass weight at 300 mg/kg bw/day,
- the NOAEL for embryo-fetal development was considered to be 100 mg/kg bw/day, based on a non statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations at 300 mg/kg/day (the increasing in fetal variations was considered to be of minor toxicological significance).

4-Tert Butylpyrocatechol did not elicit any teratogenic potential.
Executive summary:

The objective of this prenatal development toxicity study (2013) was to evaluate the potential toxic effects of the test item, 4-Tert Butylpyrocatechol, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.), inclusive).

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, 4‑Tert Butylpyrocatechol (batch No. C2262), once daily from day 6 to day 20 p.c., by gavage at dosages of 30, 100 or 300 mg/kg bw/day. An additional group of 24 time-mated females received the vehicle, corn oil, under the same experimental conditions and acted as the control group.

A dose volume of 5 mL/kg/day was used.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on clinical signs, deaths, decreased body weight and decreased food consumption, and decreased mean carcass weight at 300 mg/kg bw/day,

.         the NOAEL for embryo-fetal development was considered to be 100 mg/kg bw/day, based on a non‑statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations at 300 mg/kg/day (the increasing in fetal variations was considered to be of minor toxicological significance).

 

4-Tert Butylpyrocatechol did not elicit any teratogenic potential.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 15 June 2017 to 22 september 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
> Animal Identification: each animal was identified by tattoo in the ear.

> Environmental Acclimation: the animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

> Selection, assignment, replacement, and disposition of Animals: one day after receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the mean per subgroup. Females which were mated on the same day were classified in the same subgroup.

> Housing: following randomization, females were housed individually in cages with perforated floors equipped with water bottles.

> Environmental conditions: target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air were maintained in the animal rooms.

> Food: pelleted diet for rabbits was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay and wooden sticks were provided during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

> Water: municipal tap water was freely available to each animal via water bottles/containers. Periodic analysis of the water was performed. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

> Animal enrichment: animals were provided with shelters.

> Veterinary care: veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS AND STORAGE:

> Preparation: The dosing formulations were prepared daily and dosed within 5 hours after adding the vehicle to the test item.
> No adjustment was made for specific gravity of the vehicle and test item.
> No correction was made for the purity/composition of the test item.
> Storage: Test item dosing formulations were kept at room temperature until dosing.
> The dosing formulations were stirred continuously during dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis once on 7 August 2017 as following:
> Concentration: all groups
> Homogeneity: groups 2 and 4

All samples were stored on dry ice immediately after sampling. All samples to be analyzed were shipped on dry ice to the analytical laboratory. Upon receipt at the analytical laboratory, the samples were stored in the freezer ≤ -70 °C until analysis.

Analyses were performed by using a validated analytical procedure. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.

Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions as those used in the present study

Details on mating procedure:
> The females were purchaed timed pregnant and were arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating)
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum
Frequency of treatment:
Daily from Day 6 to Day 28 post-coitum
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose volume = 2 mL/kg
Dose concentration = 0 mg/mL
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose volume = 2 mL/Kg
Dose concentration = 5 mg/mL
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose volume = 2 mL/Kg
Dose concentration = 15 mg/mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4
Dose volume = 2 mL/kg
Dose concentration = 50 mg/mL
No. of animals per sex per dose:
22 females per dose
Control animals:
yes
yes, concurrent vehicle
Details on study design:
> Dose selection rationale: The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of the dose range finder
Maternal examinations:
> CAGE SIDE OBSERVATIONS: Yes
Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

> DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy. During the dosing period, these observations were performed at least after dosing.

> BODY WEIGHT: Yes
Animals were individually weighed on Days 2, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

> FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

> WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 post-coïtum or within 24 hours for females with abortion or early delivery
- All animals (including animals found dead or sacrificed before planned necropsy and females with abortion or early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed. No tissues, except for the uterus, were weighed.
Ovaries and uterine content:
Each ovary and uterine content of all animals was dissected and examined as quickly as possible to determine:
- Number of corpora lutea
- Gravid uterus weight
- Number and distribution of live and dead fetuses
- Number and distribution of embryo-fetal deaths
Fetal examinations:
> Live fetuses were euthanized.
Fetuses of animals found dead or sacrificed before planned necropsy were externally examined in detail and euthanized (if necessary).

> External examinations:
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight (not for fetuses of animals found dead or sacrificed before planned necropsy) was determined.
For late resorptions and recognizable fetuses of females found dead or were euthanized in extremis, a gross external examination was performed.

> Visceral examinations:
All fetuses were sexed and examined for visceral anomalies. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.

> Skeletal examinations:
The skeletal examination was done on all fetuses from all groups.

> Head examinations:
The heads were removed from approximately one-half of the fetuses for soft-tissue examination of all groups.
The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.

Parametric test: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-parametric test: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Historical control data:
The laboratory has historical data on the background incidence of fetal malformations and developmental variations in the species used (same strain and source).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
> For clinical signs noted for unscheduled deaths at 100 mg/kg, see the section "Mortality"

> For animals surviving until scheduled necropsy:
- Reduced feces production noted for animals of all groups, including controls, with a trend for increased incidence and severity with increasing dose (moderate degree reduction at dose levels up to 30 mg/kg, and up to severe degree reduction at 100 mg/kg). This finding is correlated to the reduced food consumption and of a transient nature, therefore it is considered to be non-adverse.
- Test item-related clinical signs were noted at dose levels of 30 and 100 mg/kg: grey discolouration of the mouth (one 30 mg/kg female and six 100 mg/kg females) and a lean appearance.
- For the control group laboured respiration was noted on two consecutive days for one animal (no. 4).
- All other clinical signs noted during the treatment period were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment related unscheduled deaths were noted at 100 mg/kg.
> One female at 100 mg/kg was found dead in the morning of Day 11 post-coitum before dosing (severe reduction of feces production and food consumption, 14% body weight loss, dark red contents of the stomach noted at necropsy).
> Three females at 100 mg/kg were euthanized in extremis on Day 15 post-coitum: For the three females: severe reduction of feces production and food consumption, 8-19% body weight loss / For one female: perforation in the forestomach, and grown liver and stomach, and distended caecum with gas (peforation possibly caused by a complication during the dosing procedure, which may also have caused the liver to grow together with the stomach). This complication may have added to the deteriorating state of the female.
> One female at 100 mg/kg was euthanized in extremis on Day 20 post-coitum: excessive salivation, labored respiration, rales, piloerection, pale and lean appearance, severe reduction of feces production and food consumption and body weight loss, red fluid on the manure tray, emaciation and many dark red foci in the stomach.

No mortality occurred in the 10 mg/kg and 30 mg/kg groups.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
> Body weight loss was noted for almost all females at 30 mg/kg and all females at 100 mg/kg on Day 9 post-coitum, when compared to start of treatment on Day 6 post-coitum (mean body weight loss was 4% and 8%, respectively). Body weights increased from Day 12 post-coitum onwards, but body weight gain remained statistically significantly reduced, when compared to controls, up to Day 24 post-coitum for these females at both dose levels.
> In addition, body weight gain corrected for gravid uterus was dose-dependently lower for the females at 30 and 100 mg/kg, when compared to controls (but without reaching statistical significance): mean corrected body weight gain was -64.3 gram (-1.6%) in controls, -69.9 gram (-1.8 %) at 10 mg/kg, -145.4 gram (-4.0%) at 30 mg/kg and -169.2 gram (-4.7%) at 100 mg/kg. These changes were only minimal and remained within the normal range .
> Body weights, body weight gain and body weight gain corrected for gravid uterus of animals treated at 10 mg/kg remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
> Food consumption before or after correction for body weight was statistically significantly lower over Days 6-9 post-coitum for females at 30 mg/kg and over Days 6-12 post-coitum for females at 100 mg/kg.
> Average relative food consumption values were 38% and 7% of controls, for 30 and 100 mg/kg respectively on Days 6-9 post-coitum.
> No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females at 10 mg/kg.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
> Macroscopic findings noted for unscheduled deaths are described in the section "Mortality".

> In the animals surviving until scheduled necropsy, macroscopic observations at necropsy did not reveal any alterations considered toxicologically relevant.
- Black discoloration of the skin at the lip region was noted for one female at 30 mg/kg (correlating with the grey discoloration of the mouth that was noted during in-life).
- Incidental findings were noted among control and treated animals: reddish foci located on the lungs (two control females), a wound in the skin of the foreleg (one female at 10 mg/kg), grey-white foci on the liver (one female at 100 mg/kg). Since these are occasionally seen among rabbits used and at the incidences observed in absence of a dose-relationship, they were considered to be of no toxicological significance.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): One control female delivered her offspring early on Day 26 post-coitum. No clinical signs were noted for this animal prior to early delivery, and food intake and body weight were normal. At necropsy, reddish foci were noted in the lungs. No abnormalities were noted for the fetuses.
As this occurred in a single control female only, this early delivery was not related to treatment with the test item.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment- elated effect observed.
The numbers of pregnant females in the control and test groups were similar and in the range of normal biological variation.
In total, 8 females across the dose groups were not pregnant, based on no visually observed implantation sites at necropsy: one control female, three females at 10 mg/kg, two females at 30 mg/kg and two females at 100 mg/kg. Based on the absence of a dose-relationship and as treatment started on Day 6 post-coitum, this was considered to be unrelated to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weights were considered to be unaffected by treatment.
Mean combined (male and female) fetal body weights were 39.0, 38.9, 37.4 and 38.0 grams for the control, 10, 30 and 100 mg/kg groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 100 mg/kg.
Mean sex ratios (males:females) were 49.7:50.3, 46.6:53.4, 52.1:47.9, 46.3:53.7 for the control, 10, 30 and 100 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 8.6, 9.1, 9.4 and 9.0 viable fetuses/litter for the control, 10, 30 and 100 mg/kg groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examinations were conducted for all fetuses available for fetal morphological examination.
The number of fetuses (litters) examined for this endpoint were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively.

There were no treatment related effects on external morphology following treatment up to 100 mg/kg/day. All the below malformations occurred singly in different dose groups, no dose response relationship was noted and no malformations occurred in the high dose group. These were therefore considered chance findings and were not related to treatment.

In total five externally malformed fetuses were observed: three in Group 2 (10 mg/kg) and one in Group 1 (control) and Group 3 (30 mg/kg).
> GROUP 3
The fetus at 30 mg/kg had flexure of both tarsals without underlying skeletal abnormality.
> GROUP 2:
One fetus at 10 mg/kg also had flexure of both tarsals without underlying skeletal abnormality. In addition this fetus had carpal flexures whereby the radius appeared to be missing. Besides, this fetus had several other external malformations, namely acrania and thoracoschisis and was viscerally totally malformed.
One other malformed Group 2 fetus had an omphalocele.
The last other malformed fetus at 10 mg/kg had gastroschisis, and also had a diaphragmatic hernia and small eye viscerally and sternoschisis skeletally.
> GROUP 1 (control):
The malformed control fetus had a distended abdomen that was confirmed viscerally by ascites and that appeared to have a cardiac cause (missing atrioventricular valve and large atrium). Moreover, both testes of this fetus were malpositioned.

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examinations were conducted for all fetuses available for fetal morphological examination.
The number of fetuses (litters) examined for this endpoint were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively.

There was a statistically significant increase of the litter incidence of fetuses with 13th full ribs and caudal shift of pelvic girdle in the high dose group. These respective variations were observed at an incidence of 37.8%, 44.0%, 55.6%, 76.7% and 14.5%, 14.6%, 23.9%, 38.9% per litter in Groups 1, 2, 3, and 4, respectively.
The incidences in Group 3 were above the historical control mean values (48.9% and 16.9 % per litter for 13th full ribs and caudal shift of pelvic girdle, respectively).
For Group 4 the incidences were higher than the historical control maximum values (71.4% and 38.5% per litter for 13th full ribs and caudal shift of pelvic girdle, respectively).

Although, both these skeletal variations were considered to be treatment related at 30 and 100 mg/kg, these were considered to be non-adverse since these are commonly occurring variations, and these variations are considered to have no significant biological effect on animal health or body conformity and represent slight deviations from normal).
For Group 4, the variations noted in the 13th full ribs and caudal shift of pelvic girdle resulted in a statistically significant increase in the percentage of litters with a skeletal variation and the total percent per litter with variations.
Other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Skeletal malformations in fetuses of which the dam received test item were costal cartilage anomaly (one Group 2 fetus ), rib anomaly (one Group 3 fetus), vertebral anomaly with or without associated rib anomaly (three Group 2 fetuses and two Group 4 fetuses), vertebral centra anomaly (one Group 4 fetus), sternoschisis (one Group 2 fetus) and sternal anomaly (one Group 3 fetus).
Because these malformations occurred singly, in concurrent controls (costal cartilage anomaly and vertebral anomaly with associated rib anomaly) and/or were noted previously in historical controls and no clear dose response relationship was noted, they were not considered to be treatment related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examinations were conducted for all fetuses available for fetal morphological examination.
The number of fetuses (litters) examined for this endpoint were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively.

There were no treatment related effects on visceral morphology following treatment up to 100 mg/kg/day.

Visceral malformations occurred in 4 (4), 2 (2), 5 (5) and 4 (2) fetuses (litters) in Groups 1, 2, 3, and 4, respectively.
Three out of the four affected Group 4 fetuses had a fistula and two of these fetuses had distended intestine. Historical control data include one case of a fistula in one litter and no cases of distended intestine. However, as the above two malformations occurred in one litter only, it was considered that there was a hereditary origin and that they were not toxicologically relevant.
The other malformation at the high dose was tetralogy of Fallot which was also observed in Group 3. Because this is not a rare malformation among historical controls and fell within historical control data, it was considered to be a chance finding.
In Group 3 two fetuses in two litters had intestine-diverticulum, since this malformation was observed in the absence of a dose response relationship, it was considered to be a chance finding.
The remaining visceral malformations that were observed in this study, occurred singly, and/or were noted previously in historical controls and therefore do not indicate a relationship to treatment.
All the variations noted, were not considered treatment related as they occurred infrequently, in control fetuses only, and/or occurred at frequencies that were within the range of available historical control data.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
SOFT TISSUE CEPHALIC EXAMINATION
The numbers of fetuses (litters) available for morphological examination were 172 (20), 172 (19), 188 (20) and 135 (15) in Groups 1, 2, 3, and 4, respectively. Soft tissue cephalic examination was done for approximately half of these fetuses for all groups. Since this effect have been observed in the control group and was not dose related, this effect was not considered to be related to the treatment.

No treatment- related effect observed.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No treatment related effect observed.
Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse developmental toxicity related to the treatment was observed at all dose groups.
Key result
Developmental effects observed:
no
Conclusions:
Based on the results in this prenatal developmental toxicity study, no maternal toxicity was observed in the 10 and 30 mg/kg groups, and no adverse developmental toxicity was observed at all dose levels. The malformations noted in this study were considered to be not related to the substance.
The No Observed Adverse Effect Level (NOAEL) for 4-Tert-Butylpyrocatechol were established as:
Maternal NOAEL: 30 mg/kg/day
Developmental NOAEL: at least 100 mg/kg/day
Executive summary:

The design of this study is based on the guideline OECD 414, Prenatal Developmental Toxicity Study. The objectives of this study were to determine the potential of 4-Tert-Butylpyrocatechol to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum. The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of the dose range finder.

 

At 100 mg/kg, four females were euthanizedin extremis, due to their deteriorating physical condition, and one female was found dead on Day 11 post-coitum. All five females showed severely reduced food consumption, with consequent moderate to severe body weight losses. One female showed excessive salivation, rales and labored respiration . At necropsy, emaciation and many dark red foci in the stomach were observed for this animal. For the female found dead, dark red contents of the stomach were noted at necropsy. No other signs or macroscopic findings supporting the deteriorating condition of these animals were observed. Based on the severely reduced food consumption and body weight losses, all these decedents were considered to be related to treatment with the test item. Food consumption before or after correction for body weight was statistically significantly lower over Days 6-9 post-coitum for females at 30 mg/kg and over Days 6-12 post-coitum for females at 100 mg/kg. Average relative food consumption values were 38% and 7% of controls, for 30 and 100 mg/kg respectively on Days 6-9 post-coitum. Body weight loss related to the reduced food consumption was noted for almost all females at 30 mg/kg and all females at 100 mg/kg on Day 9 post-coitum, when compared to start of treatment on Day 6 post-coitum (mean body weight loss was 4% and 8%, respectively). Body weights increased from Day 12 post-coitum onwards, but body weight gain remained statistically significantly reduced, when compared to controls, up to Day 24 post-coitum for females at both dose levels. 

As towards the end of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse. No clear test item related, adverse macroscopic findings were noted at necropsy.

No maternal toxicity was observed in the 10 and 30 mg/kg groups. Based on these findings a NOAEL of 30 mg/kg/day was identified for maternal toxicity

No adverse developmental toxicity was observed at all dose levels. The treatment related increased incidence of fetuses with13thfull ribs and a caudal shift of pelvic girdle at 30 and 100 mg/kg were considered non adverse since these are commonly occurring variations, and these variations are considered to have no significant biological effect on animal health or body conformity and represent slight deviations from normal.

No adverse treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. number of pregnant females, corpora lutea and implantation sites, or pre- or post-implantation loss,litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations) were noted up to 100 mg/kg. Based on this study the NOAEL for developmental effects is at least 100 mg/kg/day.

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Level (NOAEL) for 4-Tert-Butylpyrocatechol were established as:

Maternal NOAEL: 30 mg/kg.

Developmental NOAEL: at least 100 mg/kg

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Two reliability 1 studies according to the OECD 414 guideline are available (one on rats and the other one on rabbits)
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

Two OECD 414 test guideline studies with reliability 1 have been performed. One on rats (2013) and the other one on rabbits (2017).

The objective of this prenatal development toxicity study (2013) was to evaluate the potential toxic effects of the test item, 4-Tert Butylpyrocatechol, on the pregnant female rats and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.), inclusive). Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, 4‑Tert Butylpyrocatechol, once daily from day 6 to day 20p.c., by gavage at dosages of 30, 100 or 300 mg/kg bw/day. An additional group of 24 time-mated females received the vehicle, corn oil, under the same experimental conditions and acted as the control group.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

On the basis of the results obtained in this study:

- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg bw/day based on clinical signs, deaths, decreased body weight and decreased food consumption, and decreasedmean carcass weightat 300 mg/kg bw/day,

The NOAEL for embryo-fetal development was considered to be 100 mg/kg bw/day,  based on a non‑ statistically significant increased number of dead fetuses and a decreased fetal weight associated with increased fetal variations at 300 mg/kg/day (the increasing in fetal variations was considered to be of minor toxicological significance).

 Based on the test conditions, 4-Tert Butylpyrocatechol did not elicit any teratogenic potential in rats.

The potential of 4-Tert-Butylpyrocatechol to induce developmental toxicity after maternal exposure during the critical period of organogenesis was also investigate in an OECD 414 study in rabbit. Time-mated female New Zealand White rabbits were exposed orally by gavage to 4-Tert-Butylpyrocatechol from day 6 to 28 post-coitum, inclusive. The dose levels selected were 0, 10, 30, 100 mg/kg/day. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of fetuses (live and dead), early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

At 100 mg/kg, four females were euthanizedin extremis, due to their deteriorating physical condition (threeon Day 15 post-coitum and one on Day 20 post-coitum), and one female was found dead on Day 11 post-coitum. All five females showed severely reduced food consumption, with consequent moderate to severe body weight losses (8-19%) prior to early sacrifice or death. 

Reduced feces production was noted for animals of all groups, including controls, with a trend for increased incidence and severity with increasing dose. Feces production was up to moderate degree decreased at dose levels up to 30 mg/kg, and up to severe degree reduced at the highest dose level of 100 mg/kg. Food consumption before or after correction for body weight was statistically significantly lower over Days 6-9 post-coitum for females at 30 mg/kg and over Days 6-12 post-coitum for females at 100 mg/kg. Average relative food consumption values were 38% and 7% of controls, for 30 and 100 mg/kg respectively on Days 6-9 post-coitum. Body weight loss related to the reduced food consumption was noted for almost all females at 30 mg/kg and all females at 100 mg/kg on Day 9 post-coitum, when compared to start of treatment on Day 6 post-coitum (mean body weight loss was 4% and 8%, respectively). Body weights increased from Day 12 post-coitum onwards, but body weight gain remained statistically significantly reduced, when compared to controls, up to Day 24 post-coitum for females at both dose levels. As towards the end of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse. 

No clear test item related, adverse macroscopic findings were noted at necropsy.

No maternal toxicity was observedin the 10 and 30 mg/kg groups.

No adverse developmental toxicity was observed at all dose levels.

In conclusion, based on the results in this prenatal developmental toxicity study the following No Observed Adverse Effect Level (NOAEL) for 4-Tert-Butylpyrocatechol were established as:

Maternal NOAEL: 30 mg/kg.

Developmental NOAEL: at least 100 mg/kg

Based on the test conditions, 4-Tert Butylpyrocatechol did not elicit any teratogenic potential in rabbits.

 

 


Justification for classification or non-classification

Based on the two OECD 414 test guideline studies, one performed on rats (2013) and the second conducted on rabbits (2017), TBC is not considered as teratogen and no classification is required for this endpoint.

Despite reproductive toxicity alerts in rodent repeated-dose toxicity studies, there is no sufficiently clear elements warranting classification of 4 -tert butylpyrocatechol for reproductive toxicity (fertility), according to the criteria of CLP Regulation. An OECD 443 study has been proposed as a testing proposal in order to clarify the effect of TBC on fertilty.