oligomeric reaction product of 2-hydroxybenzaldehyde with 1-chloro-2,3-epoxypropane and phenol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 8, 2016-May 27, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

EC Number:

942-639-6

IUPAC Name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

Test material form:

solid

Details on test material:

Name: EPICLON EXA-7250
Batch/Lot number: B006
Appearance: Brown solid (in smaller amount colour is yellow)
Purity: >99%
Manufacture date: 02 October 2014
Expiry date: 02 March 2017
Storage condition: Room temperature 15-25°C, below 70 RH%
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face
mask) for unknown materials were applied to assure personnel
health and safety.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: (P) x 10 weeks old
- Weight at study initiation: (P) Males: 359.4 to 429.2 g; Females: 198.5 to 276.6 g
- Fasting period before study: No
- Housing:Polycarbonate cages (maximum [outside] dimension: 257W × 387D × 197H mm, minimum [inside] dimension: 220W × 380D × 183H mm, Tokiwa Kagaku Kikai Co.,Ltd.)
- Diet (e.g. ad libitum):Ad libitum
- Water (e.g. ad libitum):Ad libitum
- Acclimation period:21 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.1°C to 22.7°C
- Humidity (%):47.0% to 72.7%
- Air changes (per hr):6 to 20 times per hour with all fresh air
- Photoperiod (hrs dark / hrs light):12 hours per day (7:00 to 19:00)
IN-LIFE DATES: From: February 10th 2016 To: April 20th 2016

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):All diet preparation (1,200, 3,600, and 11,000 ppm) used at the initiation of administration. Stability of the test material in diet preparations had been confirmed for up to 6 weeks (42 days) refrigerated storage in a previous study.
- Mixing appropriate amounts with: The test substance was put in a container and heated up in hot water at about 70°C and then the basal diet of a 10 times amount of the test substance was gradually added while mixing the test substance and diet.
- Storage temperature of food: Diet was stored in a cold place (actual temperature: 4.2°C to 5.5°C).

Details on mating procedure:

- M/F ratio per cage: 1:1 during the mating period
- Length of cohabitation: Males and females of the same group were cohabited day and night on one-to-one basis from around 16:00 on Day 14 (first day of cohabitation) for up to 14 days.
- Proof of pregnancy: Copulation was confirmed by the presence of a vaginal plug or sperm in the vaginal smears, and the day on which the evidence of copulation was found was designated as Day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate samples were taken from the top, middle and bottom layers of each preparation of the dietary formulations and extracted into acetonitrile (1g diet extracted into 100mL acetonitrile) by ultrasonic vibration. The solution was centrifuged and an aliquot of the supernatant was diluted as appropriate in acetonitrile to give a nominal concentration of test material in solution of approximately 1µg/mL.

Sample solutions were analysed by HPLC with UV detection (230nm). Standard solutions in the range 0.5 - 2.0µg/mL were analysed concurrently with the sample solutions for calibration purposes.

The test material formulations were within the acceptability criteria (mean of the measured values should be within ±15% of the nominal concentration and the coefficient of variation of measured values should be equal to or less than 15%). Dose levels in diet and ingested by the animals are quoted in terms of the nominal concentration.

Duration of treatment / exposure:

Animals were administrated for 2 weeks before mating, through the mating period until the day of necropsy (males: 28 days in total, females: until Day 13 of lactation; 50 to 54 days in total). Non-delivering females were administrated until 26 days after copulation.

Frequency of treatment:

Continuous (test material was incorporated into the animals' diet which was made available ad libitum)

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks (animals were 10 weeks old at the initiation of administration and dosed for 2 weeks before the start of mating).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Untreated diet control group

Doses / concentrations 2

Dose / conc.:

1 200 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

3 600 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

11 000 ppm (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels (test substance concentrations) were set at 1,200, 3,600, and 11,000 ppm.
In the study entitled “A 28-Day Repeated Dose Oral Toxicity Study of EPICLON EXA-7250 in Rats —Dietary Administration—” (Study No. B150379, test substance concentrations in the diet: 0, 1,200, 3,600, and 11,000 ppm) conducted at the test facility, there were no test substance-related toxic changes in clinical signs or body weight and no macroscopic abnormalities were observed in the reproductive organs or their accessories in males and females at necropsy.
Therefore, the test substance concentration in the diet for the high dose group was set at 11,000 ppm in the same with the study entitled “A 28-Day Repeated Dose Oral Toxicity Study of EPICLON EXA-7250 in Rats —Dietary Administration—”. The test substance concentrations for the middle and low dose groups were set at 3,600 and 1,200 ppm, respectively, to assess the relationship between toxic changes and dose levels. A control group administered with the basal diet (0 ppm) alone was also established.

Examinations

Parental animals: Observations and examinations:

Clinical observation
All animals were observed every day for clinical signs from the initiation of administration to necropsy. Frequencies of the observation are listed below.
Dosing period: Twice a day (in the morning and afternoon)
Other days: Once a day

BODY WEIGHT: Yes
All animals were weighed on the days shown below with electronic balances.
Males: The day of initiation of administration and once a week until the day of necropsy (including the day of necropsy)
Females: The day of initiation of administration and once a week until the successful copulation
Days 0 (copulation), 7, 14, and 20 of gestation
Days 0 (parturition), 4, and 13 of lactation
Non-delivering females were also measured on the day of necropsy.

In addition, body weight gain was calculated on the basis of the body weight on the day of initiation of administration during the pre-mating period, Day 0 of gestation during the gestation period, and Day 0 of lactation during the lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The gross weight of the diet (including feeder) was measured with electronic balances, and the food consumption during the measurement periods shown below was calculated.
Males: The day of initiation of administration and once a week until the day of initiation of copulation. In addition, the food consumption of males, which succeeded in copulation within 8 days after the initiation of mating, was measured on Days 21 and 28 (the day of necropsy).
Females: The day of initiation of administration and once a week until the day of initiation of copulation
Days 0 (copulation), 7, 14, and 20 of gestation
Days 0 (parturition), 4, and 13 of lactation
Non-delivering females were also measured on the day of necropsy to calculate the test substance intake.
Food consumption was not measured during the cohabitation period. The mean daily food consumption was calculated based on the difference in the gross weight between the measurement days.

The test substance intake during each food consumption measurement period (see above) during the dosing period was calculated.

Oestrous cyclicity (parental animals):

The estrous cycle was examined by collecting vaginal smears every day in the morning from the initiation of administration until the initiation of mating. From the results of the examination, the mean estrous cycle length for each female and the index of females with irregular estrous cycles were calculated. Females that had an estrous cycle length other than 4 to 6 days were judged as having irregular estrous cycles.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Testis (organ weight and histopathology), epididymis (weight and histopathology), seminal vesicles / coagulating gland, prostate (ventral and dorsal lobes), cowper's gland (organ weight), and glans penis (including organ weight).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (equal sex ratio in principle); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Live birth index, viability index on day 4, viability index on day 13, external anomaly index, index of dams with external anomaly offspring, sex ratio on day 0, sex ratio on day 4.
Pup bodyweigts were recorded on postnatal days 0, 4 (before and after litter size adjustment) and 13.
Ano-genital distance was measured on postnatal day 4.
Observation of nipple development was observed in all surviving male pups on postnatal day 12.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities except for cannibalised pups

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on day 28 of dosing
- Maternal animals: All surviving animals were sacrificed on day 13 of lactation or 26 days after copulation in the event of non-delivery

Organs / tissues were examined as indicated in table 1

Postmortem examinations (offspring):

All survived offspring were observed externally including the oral cavity and external genitalia, euthanized in the same manner with parental animals, and the head, thoracic, and abdominal organs/tissues were examined macroscopically on postnatal Day 13. The thyroids of 1 male and 1 female offspring (selected in descending order) per dam were collected, fixed, and preserved in 10 vol% phosphate-buffered formalin.
The offspring that died before the litter size adjustment, except for cannibalized ones, were examined for the presence of external anomalies and the whole bodies were preserved in 10 vol% phosphate-buffered formalin. The offspring that died after the litter size adjustment was observed externally and also preserved in the same manner.

The bilateral thyroids of 1 male and 1 female offspring per dam were weighed together.

Statistics:

The data of offspring were analyzed on the basis of litter mean values. The body weight and food consumption data from non-pregnant females after copulation were excluded from the analysis.
As for body weight, body weight gain, food consumption, organ weight, relative organ weight, number of implantations, number of offspring (live and dead), AGD and TSH and Total T4 concentrations, homogeneity of the variance among the groups was first tested by Bartlett’s test. When the variance was homogeneous, the following A was performed. When the variance was heterogeneous, Bartlett’s test was applied again using the log-transformed values, and the following A was performed using the log-transformed values when the variance of the log-transformed values was homogeneous. When the variance of the log-transformed values was heterogeneous, the following B was performed using the rank-transformed values. A: One-way analysis of variance was performed. When a significant inter-group difference was found, Dunnett's multiple comparison test was performed. B: Kruskal-Wallis test was performed. When a significant inter-group difference was found, Steel multiple comparison test was performed. As for the partial reproductive and developmental data (Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, birth index, external anomaly index, live birth index, viability index on Days 4 and 13, and nipple development anomaly index), the above B was performed. As for index of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (postnatal Days 0 and 4), and index of dams with external anomaly offspring, Fisher’s exact probability test was performed.

Reproductive indices:

Live birth index(%) was calculated on postnatal days 0, 4, and 13 as the ratio of the number of offspring born alive (x100) divided by the total number of offspring born.

Offspring viability indices:

Viability index on Day 4 (%):
(Number of offspring alive on Day 4/Number of offspring born alive) × 100
Viability index on Day 13 (%):
(Number of offspring alive on Day 13/Number of offspring alive after the litter size adjustment on Day 4) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities in clinical sign were observed in males or females in the treatment groups during the experimental period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 11000 ppm group a statistically significant low body weight was noted in females throughout the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 11,000 ppm group, a statistically significant low food consumption was noted in females throughout the lactation period.
A lower food consumption in males and females in the 11,000 ppm group from Day 0 to 7 during the pre-mating period was not considered to be treatment-related because it was a transient change observed only shortly after the initiation of administration.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in the plasma TSH or Total T4 concentrations.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted in males or females, including the cecum with distention.
Besides, the histopathological findings in the testis and/or epididymis in the control and 11,000 ppm groups were not judged to be treatment-related because they are nonspecific changes sporadically observed in rats and there were no apparent differences in the incidence between the control and 11,000 ppm groups.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in the mean estrous cycle length or irregular estrous cycle.
Almost all females had a normal estrous cycle of 4 to 5 days and no significant difference was noted in the mean estrous cycle length between the control and treatment groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted in the number of days until copulation, number of estrous stages without copulation, copulation index, or fertility index.
All the mating pairs copulated during the first estrus stage of females after the start of mating. In the 1,200 ppm group, a lower number of days until copulation was noted; however, it was not considered to be treatment-related because there was no dose-dependency. There was one non-pregnant female observed in each of the control and 1,200 ppm groups; however, there was no significant difference in the fertility index between the control and treatment groups. Each one female in the 1,200 ppm group and 11,000 ppm group, which showed an irregular estrous cycle before mating, copulated during the first estrous stage after the start of mating and conceived.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

11 000 ppm (nominal)

Based on:

test mat.

Remarks:

corresponds to 672.44 mg/kg/day

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive function (oestrous cycle)

2

Key result

Dose descriptor:

NOAEL

Effect level:

3 600 ppm (nominal)

Based on:

test mat.

Remarks:

Corresponds to 334.87 mg/kg/day

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive function (oestrous cycle)

reproductive performance

3

Dose descriptor:

LOAEL

Effect level:

11 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

4

Dose descriptor:

LOAEL

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Before culling, deaths, loss of suckling, and/or bite wounds on the tail were sporadically observed in all groups; however, there were no obvious differences in the incidences among the groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Deaths were seen sporadically in all groups; however there were no obvious differences in the incidences between groups. Additionally 1 female in the control group was found dead on postnatal day 10.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 11,000 ppm group, a statistically significant low body weight with low body weight gain was noted in males and females on postnatal Day 13.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in the plasma TSH or Total T4 concentration.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related changes in the thyroid weight were noted in males or females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormalities were observed in males or females in the treatment groups. In 1 male in the control group, dark reddish patch in the lungs and brain, hemorrhage in the abdominal cavity, and anemic change in the whole body were noted.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in nipple development anomaly index.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 600 ppm

Based on:

test mat.

Remarks:

Corresponds to 334.87 mg/kg/day in maternal animals

Sex:

male/female

Basis for effect level:

clinical signs

mortality

organ weights and organ / body weight ratios

gross pathology

2

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

11 000 ppm

Based on:

test mat.

Remarks:

Corresponds to 990.81 mg/kg/day in maternal animals

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Effect was thought to be secondary to low body weight and food consumption in dams during the lactation period

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the no-observed-adverse-effect level (NOAEL) was considered to be as follows under the conditions of this study. As for the parental animals for general toxicity, NOAEL in parental males was 11,000 ppm (672.44 mg/kg/day) because no toxicological changes were observed at the high dose and NOAEL in parental females was 3,600 ppm (334.87 mg/kg/day) because low body weight and food consumption were noted at the high dose during the lactation period. NOAEL for the reproductive function of parental animals was 11,000 ppm (males: 672.44 mg/kg/day, females: 990.81 mg/kg/day) because no toxicological changes were observed at the high dose.
NOAEL for the development of offspring was 3,600 ppm (334.87 mg/kg/day) because suppressed body weight gain was noted at the high dose although it was considered to be a secondary change associated with low body weight and food consumption in dams during the lactation period.

Executive summary:

EPICLON EXA-7250 was administered orally (dietary administration) at dose levels (test substance concentration in the diet) of 0 (control), 1,200, 3,600, and 11,000 ppm to male and female Crl:CD(SD) rats to assess the reproductive/developmental toxicity of EPICLON EXA-7250. The male rats were dosed from 14 days before mating to the day of necropsy (including the mating period; 28 days in total), and the female rats were dosed from 14 days before mating to Day 13 of lactation (including the mating period, gestation period, and delivery; 50 to 54 days in total). Each test group consisted of 12 males and 12 females. The control group was given the basal diet (CR-LPF, Oriental Yeast Co., Ltd.) only. In this study, the mean test substance intakes at 1,200, 3,600, 11,000 ppm were 73.19, 217.66, and 672.44 mg/kg/day for males and 113.83, 334.87, 990.81 mg/kg/day for females, respectively.

As for the general toxicological effects on parental animals, low body weight and food consumption were noted in females at 11,000 ppm during the lactation period. At necropsy, distention of the cecum was observed in males and females at 11,000 ppm; however, this was not judged to be toxicologically significant because of the absence of the histopathological change in the cecum. There were no test substance-related toxicological changes in clinical signs, plasma TSH or T4 concentration, organ weight, or histopathological examination of the reproductive organs. As for the effects on the reproductive performance, there were no test substance-related changes in estrous cycle, mating, fertility, pregnancy, or observation of parturition or nursing. As for the effects on offspring, low body weight with suppressed body weight gain was noted in males and females at 11,000 ppm on postnatal Day 13. It was considered to be a secondary change associated with the adverse effect on dams because low body weight

and food consumption were noted in dams during the lactation period and there were no abnormalities in other parameters in offspring. There were no test substance-related toxicological changes in viability, external examination, anogenital distance (AGD), plasma TSH or T4 concentration, nipple development, thyroid weight, or necropsy.

In conclusion, the no-observed-adverse-effect level (NOAEL) was considered to be as follows under the conditions of this study. As for the parental animals for general toxicity, NOAEL in parental males was

11,000 ppm (672.44 mg/kg/day) because no toxicological changes were observed at the high dose and NOAEL in parental females was 3,600 ppm (334.87 mg/kg/day) because low body weight and food consumption were noted at the high dose during the lactation period.

NOAEL for the reproductive function of parental animals was 11,000 ppm (males: 672.44 mg/kg/day, females: 990.81 mg/kg/day) because no toxicological changes were observed at the high dose.

NOAEL for the development of offspring was 3,600 ppm (334.87 mg/kg/day) because suppressed body weight gain was noted at the high dose although it was considered to be a secondary change associated with low body weight and food consumption in dams during the lactation period.