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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes

Test animals

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.

Administration / exposure

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).

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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).

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

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

Overall developmental toxicity

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

Any other information on results incl. tables

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)).

Applicant's summary and conclusion

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.