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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional group(s) and common mechanism(s) of action.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3,5-trimethylhydroquinone
EC Number:
211-838-3
EC Name:
2,3,5-trimethylhydroquinone
Cas Number:
700-13-0
Molecular formula:
C9H12O2
IUPAC Name:
2,3,5-trimethylbenzene-1,4-diol
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Trimethylhydroquinone
- Physical state: yellow powder
- Analytical purity: 99 .8%
- Purity test date: Nov 09, 1994
- Lot/batch No.: 4361662
- Stability under test conditions:
purity and chemical identification of the test material was determined prior to the start of the study and at the end of the study.
- Storage condition of test material:
room temperature (19/08/93 to 14/09/93)
afterwards: room temperature in the dark, over
silica gel, under nitrogen gas
Specific details on test material used for the study:
Yellow powder, melting point 170,9 ℃
Purity 98,8%
Storage conditions: 4, 6 to 7.0 ° C, dark, under nitrogen
Supplier:

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Source
Charles River Japan Co., Ltd. (Atsugi Production Center)
Number of purchased animals
52 males, 62 females
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST animals:
number: 48 male, 58 female
age: 9 weeks
weight at starting administration:
male: 297 to 368 g,
female: 190 to 230 g

Diet:
Radio-sterilized solid feed for experimental animals (CRF-1, Oriental Yeast Co., Ltd.), ad libitum
Water: ad libitum

Acclimation period: 1 week

Identification of anmimals: labelled on tails, after grouping by ear punching

Environmental conditions
Temperature
21.4 to 22.2 ° C (tolerance range 19.0 to 25.0 ° C)
21.2 to 23.3 ° C (tolerance range 19.0 to 25.0 ° C)
Relative Humidity
49.4 to 64.3% (tolerance range 35.0 to 75.0%)
44.9 to 61.1% (tolerance range 35.0 to 75.0%)
Ventilation
6 to 20 times / hour, fresh air supply
Photoperiod:
12 hours / day (7: 00-19: 00)

Cage
(1) Period excluding the whole period of male and female pregnancy / nursing period
Stainless steel hanging wire mesh cages (195W × 325D × 180H mm), exchanged once within 15 days.
(2) pregnancy / nursing period
Using a polycarbonate cage (265 W × 426 D × 200 H mm), exchanged once within 7 days.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: (0.5 w / v% CMC-Na containing 0.1 w / v% Tween 80)
Details on exposure:
female:
14 days before mating, mating period, gestation period and 4 days of nursing through delivery (day of delivery = nursing day 0)
male:
14 days before mating, and a total of 42 days from the mating period to the day before necropsy.
A recovery period of 14 days after the end of the administration period was given to 5 males and 5 females in the control and in 300 mg / kg group.
Details on mating procedure:
mating period up to 14 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
By HPLC method for each dose. The average value of each concentration is within the set concentration ± 10%, C.V. value is within 10% (upper layer, middle layer, lower layer) of the sample. Set concentration of each administration solution is 95.8 to 98.7%, C.V. value is 0.5 to 0.6% (see Attachment 12.4).
Duration of treatment / exposure:
Male
A total of 42 days were taken from 14 days before mating and through the mating period to the day before necropsy.
Female:
42 - 56 days
Pregnant and lactating animals
14 days before mating, mating period, gestational period, and 4 days of lactation through parturition day. (Day 0) However, females who did not mate were kept until the day before necropsy.
Females who did not copulate were treated to the day before necropsy.
Frequency of treatment:
once a day between 8:28 and 12:16.
Details on study schedule:
Start of test December 9, 2005
Start of experiment December 14th, 2005
Animal arrival December 14, 2005
Administration December 22, 2005 - February 15, 2006
Mating period starting January 5 th, 2006
Parturition January 28th to February 11th 2006
Nursing 4 day until neonatal dissection February 1, 2006 to February 15, 2006
Male dissection after the administration period February 2nd, 2006
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
recovery
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Remarks:
recovery
No. of animals per sex per dose:
12 per sex per dose
Control animals:
yes
Positive control:
no

Examinations

Parental animals: Observations and examinations:
behaviour,
body weight, food consumption,
hematology, blood biochemistry,
male urinanalysis,
Organ weight
pathological examination (anatomic alterations, histopathology
Oestrous cyclicity (parental animals):
cytological examination
Sperm parameters (parental animals):
histopathological examination of testis and epididymis
Litter observations:
number of birth, presence of sex and outer abnormalities, body weight,
Postmortem examinations (parental animals):
yes
Postmortem examinations (offspring):
no
Statistics:
yes
Reproductive indices:
gestation index, number of corpora lutea, number of implantation sites, number of pups
Offspring viability indices:
number of living pups on day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation (300 mg/kg bw), Irregular respiration (300 mg/kg bw),
Decrease in locomotor activity (300 mg/kg bw), Tonic convulsion (agonal stage), Clonic
convulsion (agonal stage)
Mortality:
mortality observed, treatment-related
Description (incidence):
300 mg/kg bw
Male death 3 of 12
Female 7 of 12
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
M, F slightly decreasing 300 mg/kg bw
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
M, F slightly decreasing 300 mg/kg bw
In the 300 mg / kg group, no statistically significant difference was found. However, in female experimental animals, the tendency to low levels was noted from the 14th to the 20th day of gestation.
The female test animals of the 300 mg / kg satellite group showed a significantly high value on the 36th day. But since the change was only temporary, it was considered a coincidence. In the male experimental animals of the 10 mg / kg group and female animals of the 60 mg / kg group no significant differences to the control group were found.
Water consumption and compound intake (if drinking water study):
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the application phase, low levels of red blood cells, hemoglobin concentration and hematocrit, as well as high levels of platelets and reticulocytes, were observed in male animals of the 300 mg / kg group. In addition, low values ​​of red blood cells, hemoglobin concentration, and hematocrit, and a tendency for platelets and reticulocytes to be high, were measured in male animals of the 60 mg / kg group. Further, there was a tendency for high levels of leukocytes in both male and female experimental animals of the 300 mg / kg group. At the end of the regeneration phase, low erythrocyte values ​​and high levels of reticulocytes were measured in male animals of the 300 mg / kg group. In addition, a low value of mean corpuscular hemoglobin concentration and a longer thromboplastin time were measured in female experimental animals of the 300 mg / kg group. However, since the difference was marginal and no change in male experimental animals of the same group could be detected at the end of the application phase, this is considered a coincidence.
In male animals of the 10 mg / kg group and in female animals of the 10 and 60 mg / kg group no significant difference to the control group was found.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After the end of the application phase, a high level of total bilirubin was measured in female experimental animals of the 300 mg / kg group. In addition, although a high level of ALP activity was measured in female animals of the 60 mg / kg group, no difference was found in the 300 mg / kg group and no compound was found, therefore this is considered a coincidence. Although the study at the end of the regeneration phase found a high A / G ratio in female animals of the 300 mg / kg group, no difference was found in the study after the end of the application period in male experimental animals, so this was considered as coincidence.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Male: Protein, glucose, Ketone body, Bilirubin, Urobilinogen were tested
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the planned autopsy of the experimental animals changes in the spleen and the liver were found in both male and female experimental animals, which suggest a effect of the test substance.
In 1 male test animal of the 60 mg / kg group, in 5 male and 4 female experimental animals of the 300 mg / kg group, respectively after the application phase were autopsied and in 4 male animals of the 300 mg / kg group, according to the Regeneration phase were autopsied, elevated levels of hemosiderin dye were detected in the spleen. Hemosiderin deposits were found in 5 male and 3 female animals of the 300 mg / kg group, respectively, after the application phase, and in 3 male and 4 female animals of the 300 mg / kg group, which were postoperatively post-recovery detected on the Kupffer cells of the liver. In 1 female test animal of the 60 mg / kg group, which was autopsied after the regeneration phase, a subcutaneous tumor was detected. Histologically, it was adenocarcinoma of the mammary gland. In 1 female test animal of the 300 mg / kg group, which was autopsied after the regeneration phase, abnormal lobation of the liver was noted. Histologically, however, it was normal liver tissue. Inflammatory infiltration of the adrenal zona reticularis was detected in 2 female animals of the 300 mg / kg group and 1 female animal of the 10 and 60 mg / kg groups. In addition, various histological changes in dead animals, in experimental animals that were autopsied after the application phase and in experimental animals that were autopsied after the regeneration phase, found. However, these changes occurred unspecifically and since their occurrence was not clearly group-dependent, they are assessed as not dependent on the test substance. Furthermore, no abnormalities were found in the ovaries of non-pregnant animals.

Deceased animals of the 300 mg / kg groups were found to have degenerations of the cardiac muscle 1 case of middle and heavy degree in male animals and 1 case of slight degree in a female animal. In 2 male experimental animals, advanced extramedullary hematopoiesis of erythrocytes in the spleen was found in 2 male 5 female experimental animals increased levels of hemosiderin dye also in the spleen. In 3 male and 1 female experimental animals, some eosinophilic inclusion bodies were detected in liver cells. In 3 female animals slight hemosiderin deposits were detected on the Kupffer cells. In addition, medium-grade erosion in the anterior stomach area was observed in 1 male test animal, slight uterine and vaginal bleeding in 2 female test animals, and inflammatory infiltration of the adrenals zona reticularis in 1 female test animal. In 2 female experimental animals pulmonary edema and in 1 female experimental animal anomalous contents in the stomach and the ileum, but no histological changes, were found. In addition, although changes were detected in the lung, kidney, thyroid and parathyroid glands, these were also detected nonspecifically in the control group and thus suggest that they are not related to the test substance.

In the dam, of which all descendants have died, degeneration of the mid heart muscle, advanced extramedullary hematopoiesis of erythrocytes and eosionophilic inclusion bodies in liver cells has been noted. This corresponded to the findings in other dead animals. In addition, focal necrosis of liver cells, extramedullary hematopoiesis, mammary atrophy, pulmonary edema, blood clots, erosion of the duodenum, inflammatory infiltration of the uterine and vaginal mucosa were noted.
As described, mild to severe degeneration of the heart muscle has been noted in dead animals and in the dam, whose entire offspring have died. However, post-mortems after the application phase were
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
In the study of the oestrus cycle, no change in the average value of the respective groups was found and thus no change that could be attributed to the test substance. Also, there were no experimental animals with abnormal oestrus cycles. As a result of mating, all trial pairs have mated up to 1 trial pair of the 300 mg / kg group. At the mating rate, days to mating, missed estri, as well as conception rate, no significant difference between application group and control group was found.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In the 300 mg / kg group, from the 15th to the 22nd day of pregnancy, 4 out of 10 experimental animals died and led to a reduction in the birth rate. The deceased animals were examined for number of corpus luteum and implantations. Thus, a implantationrate could be determined, which flowed into evaluation. As a result, low values of corpus luteum could be detected in the same group, indicating a tendency to low levels of implantations and offspring. With gestation time, implantationrate and total birth rate no significant difference could be determined between application group and control group.
Although dying of all offspring of 1 dam of the 300 mg / kg group was observed on day 1 of the nursing phase, no abnormal lactation was observed. In other dams, no abnormal behavior was observed at birth or breastfeeding.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related

Details on results (F1)

The number of live male offspring 4 days after birth in the 300 mg / kg group was low. Also, a tendency to low levels of total 4-day survival animals and survival was noted. In the general findings, as well as the external examination of the offspring no anomalies could be determined on the basis of the test substance.
Furthermore, a deviation from the control group in the sex distribution was found in the 60 mg / kg group, but since such a deviation could not be detected in the 300 mg / kg group, this is considered a coincidence.
Body weight
See Table 38, Appendices 41, 44!
The body weight could not be significantly different from the control group in either male or female animals.
Autopsy
See Table 40, Appendix 43!
In the autopsy of fourth offspring, non-breastfeeding or cannibalism was sporadically detected in the various groups, but no abnormalities attributable to the test substance.

Effect levels (F1)

Key result
Generation:
F1
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not measured/tested
Remarks:
acc. to the OECD Guideline 422 derivation of effect levels for F1 is not foreseen and du to the limits of study design not possible.

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Since the increase in spleen hemosiderin pigment was observed in the group of 60 mg / kg or higher, in females death and increase in spleen hemosiderin pigment was observed in 300 mg / kg group, NOEL for reproductive and developmental toxicity is considered to be 60 mg/kg/day.
In animals, the birth rate, the low value of the corpus luteum, and the low value for the death of all newborns and the survivability of newborns. It is considered to be 60 mg / kg / day.
NOEL on Reproductive Developmental Toxicity and NOAEL was unchanged in male animals at 300 mg/kg /day, dams and F1 children.
In animals, the birth rate, low number of corpora lutea and low values for all-birth mortality and neonatal survival the NOAEL is considered to be 60 mg/kg.
Executive summary:

4 out of 10 animals in the 300 mg / kg group died during pregnancy (15-22 days gestation) and this resulted in a low birth rate. In addition, 1 female died from the group during the nursing phase. Also, low levels of corpora lutea were noted, and thus a tendency to low levels of implantation and live births. The female experimental animals showed no abnormalities in general findings, body weight or feeding quantity before the mating phase. A negative effect of the test substance on the stated values during ovulation (14-21 days of application) does not seem likely. Therefore, it is assumed that the test substance has caused a reduction of the oocytes (low values of corpora lutea). A reduction of implantations relative to corpora lutea or live births to implantations could not be detected, thus excluding negative impact on implantation or pregnancy. During the nursing phase, 1 case occurred in which offspring of a dam of the 300 mg / kg group died. Normal nursing behavior was observed on the day of birth, but it is believed that at this time the mother already suffered from circulatory problems due to myocardial degeneration, so that it is assumed that the situation thereafter rapidly deteriorated,

resulting in impaired breastfeeding ability and thus death of all descendants resulted. Further changes in oestrus cycle, mating rate, conception rate, gestation time or birth due to the test substance could not be determined. The deaths of all offspring of a dam lead to low values of non-dead offspring and the 4 -day survival rate. Other changes in live birth rate, general findings, examination of body surface area, body weight and autopsy, which could be attributed to the test substance, could not be determined.

This gives the following NOEL and NOEAL for 2,3,5-trimethylhydroquinone under the given experimental conditions. The NOEL and NOEAL for repeated-dose toxicity in males is 10 mg / kg / day because of death in the 300 mg / kg group and elevated levels of hemosiderin dye in the spleen in the 60 mg /kg group were found. In females, the value is 60 mg / kg / day since deaths and increased levels of hemosiderin dye in the spleen were noted in the 300 mg / kg group. Concerning. Reproductive toxicity of NOEL and NOEAL in males is 300 mg / kg / day, as no change could be detected. In dams and the F1 generation, the value is 60 mg / kg / day as low rates of birth rate, corpus luteum, survivability of

offspring and one death of all offspring of a dam were found.