1,3,5-tris[[4-tert-butyl-3-hydroxy-2,6-xylyl]methyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Results from an OECD 421 reproductive/developmental toxicity screening study in rats

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

9 June 2004 to 22 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

2003-02-13

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley Cr1:CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent
- Weight at study initiation: Males: 340-394 g; Females: 209-243 g
- Fasting period before study: none
- Housing: Initially housed in groups of five (5) animals by sex in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During mating animals transferred to similar cages with one male and one female per cage. Following evidence of successful mating, males returned to original cages. Mated and presumed pregnant females housed individually in polypropylene cages with solid floors and stainless steel tops. Mated females given softwood chips as bedding throughout gestation and lactation.
- Diet (ad libitum): Certified Rodent Diet PMI 5002, IPS Product Supplies Ltd., London (UK)
- Water (ad libitum): mains water supplied in polycarbonate bottles
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 9 June 2004 to 22 July 2004

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION

A known amount of test compound was mixed with basal laboratory diet for twenty minutes at a constant speed (setting 1, Hobart QE800 mixer). Analytical measurements prior to the start of the study indicated the dietary mixture to be homogenous and stable for at least 14 days.

Details on mating procedure:

One male and one female were paired for a period of up to fourteen days. Following pairing, the polypropylene trays beneath each cage were checked each morning for the presence of ejected copulation plugs. Additionally, each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating. Mated females were separated and housed individually during the period of gestation and lactation. Males were returned to original holding cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the subject chemical in dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique. Samples were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 100 ppm. The dietary mixtures were sampled sampled and analyzed within four (4) days of preparation for verification of formulation concentrations. Measured concentrations varied from 97 to 107% of nominal.

Duration of treatment / exposure:

Test material was administered for up to 54 days by dietary admixture. Control animals were handled in the same manner to those receiving the test material.
Both male and female animals were dosed 14 days prior to pairing. Dosing continued until surviving parental animals were sacrificed on Day 5 post partum.

Frequency of treatment:

Continuous

Details on study schedule:

Administration of doses was started 14 days prior to mating. Mating was allowed to proceed for up to 14 days. Pregnant females were allowed to deliver offspring. Offspring were observed for growth and development during lactation up to Day 4 post partum. Surviving adults and offspring were euthanized on Day 5 post partum and examined macroscopically.

Dose / conc.:

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

Remarks:

Males 1000 ppm

Dose / conc.:

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

Remarks:

Males 10000 ppm

Dose / conc.:

1 264 mg/kg bw/day (actual dose received)

Remarks:

Males 20000 ppm

Dose / conc.:

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

Remarks:

Females 1000 ppm

Dose / conc.:

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

Remarks:

Females 10000 ppm

Dose / conc.:

1 558 mg/kg bw/day (actual dose received)

Remarks:

Females 20000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the outcome of a preliminary 14-day oral gavage range-finding study.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were checked twice daily for morbidity and mortality during the normal working week and once daily on weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill health or behavioural change once daily. Abnormal findings were recorded daily until resolution of finding or study termination.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on Day 0 before start of treatments and weekly thereafter for males until termination. Females were weighed weekly until mating was evident. Parental females showing evidence of mating were weighed on Days 0, 7, 14 and 20 post coitum. Parental generation females with a live litter were weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- During the maturation period food consumption was recorded weekly for each cage of parental generation adults. For parental generation females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 20 post coitum. For parental generation females with live litters, food consumption was recorded for the period covering Days 1 to 4 post partum.
- Group mean weekly food consumption was calculated (as g/rat/day)
- Weekly food efficiencies were calculated (group mean body weight gain (as g/rat/week) / group mean food consumption (as g/rat/week))

WATER CONSUMPTION
- Time schedule for examinations: daily visual inspections of water bottles

HISTOLOGY/HISTOPATHOLOGY
The following organs were collected and preserved in buffered 10% formalin and examined at histopathology for all adult males and females from the control and high dose groups: coagulating glands; epididymides; prostate; seminal vesicles; testis; pituitary; ovaries; uterus/cervix; vagina; skin (from top of head). Skin samples from all groups were prepared as paraffin blocks, sectioned at nominal thickness of 5 mm and stained with hematoxylin and eosin for subsequent microscopic examination. All other tissues from control and 20000 ppm dose group animals were treated in similar manner.

OTHER: Each pregnant female was observed at 8:30, 12:30 and 16:30 hours at or around the period of expected parturition. On weekends, observations were carried out at 8:30 and 12:30 hours only. The following were recorded for each female:
- date of mating
- date and time of observed start of parturition
- date and time of observed completion of parturition
- duration of gestation

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

not determined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number of pups born
- number and sex of pups alive recorded daily and on Day 1 and 4 post partum
- clinical condition of pups from birth to Day 4 post partum
- individual litter weights on Day 1 and 4 post partum

All live offspring were observed for detachment of pinna (as noted by the separation of the edges and subsequent unfolding of both pinnae) and assessed for reflexological response to stimuli by assessing surfact righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Offspring dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities recorded.

Postmortem examinations (parental animals):

All adult (P) animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. In addition, corpora lutea and implantation sites of all females were counted. This procedure was enhanced by staining the uteri with 1% ammonium polysulphide solution.

Postmortem examinations (offspring):

All surviving offspint were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Group mean values and standard deviation were derived where appropriate. Weekly bodyweight, bodyweight gain, food consumption, adult organ weights, litter sizes, litter weight and offspring bodyweights were assessed for dose response relationships by analysis to establish homogeneity of group variances using Bartlett's test followed by one-way analysis of variance. If variances were unequal subsequent comparisons between control and treated groups were performed using t-test assuming unequal variances. If variances were equal subsequent comparisons between control and treated groups were performed using Dunnett's Multiple Comparison Method.

Offsping landmarks of physical development, offspring reflexological responses, pre- and post-implantation losses, litter sex ratios and relative organ weights were compared using Kruskal-Wallis non-parametric rank sum test. Post implantation losses in the 10000 ppm group were also compared using Grubb's test (parametric test to determine outliers).

For histopathology observations: Chi-squared analysis was used for differences in lesions occurring with an overall frequency of 1 or greater; Kruskal-Wallis on-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.

Reproductive indices:

Pre-coital interval: calculated as time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility indices: mating index (%) = number animals mated/number paired x 100; pregnancy index (%) = number pregnant females/number animals mated x 100.
Gestation length: calculated as the number of days of gestation including the day for observation of mating and start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day.
Parturition index (%) = number of females delivering live pups/number of pregnant females x 100

Offspring viability indices:

Live birth index (%) = number of pups alive on Day 1/number of pups born x 100
Viability index (%) = number of pups alive on Day 4/number of pups alive on Day 1 x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 20000 ppm three males and all females showed scabs and fur loss on the top of the head and/or around the snout from Day 22 of treatment onwards.

At 10000 ppm all females showed scabs and fur loss on the top of the head and/or around the snout from Day 22 of treatment onwards. For all but one female, these signs persisted until completion of the study. The skin irritation on the top of the head and/or around the snout is considered an adverse effect of treatment due to the unique nature and severity of the irritancy.

At 1000 ppm there were no clinical signs of toxicity or irritancy observed throughout the study.

One control female had approximately 2 inches of skin missing from the end of the tail exposing underlying tissue.

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related mortalities through the course of the study at any dose level.
One control female was killed in extremis due to an injury to the tail. No other macroscopic abnormalities were observed at post mortem examination.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant effects on bodyweight gain were observed for animals of either sex throughout maturation or for females during gestation or early lactation. Bodyweight development in test animals was similar to that of controls.

At 20000 ppm there was a reduction ini female bodyweight gain for females between days 14 and 20 of gestation. This resulted in a slight, but not statistically significant difference in group mean female bodyweight at day 20 of gestation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant increase in the food eaten during the first week of treatment in male rats at 20000 and 10000 ppm compared to controls. This was considered to be incidental due to the lack of a dose related response and comparable female food consumption.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Acanthosis, occasionally with focal epithelial ulceration and overlying scab formation, was observed in relation to treatment in the majority of females dosed at 20000 ppm (p<0.001) or at 10000 ppm (p<0.01). One male at 20000 ppm was also affected. Other histopathological findings were those commonly encountered as a result of pregnancy or commonly observed in laboratory rats of this age and strain.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were no apparent effects on mating performance or fertility or gestation length. At the 20000 ppm dose level, there was no evidence of mating for one mating pair but a live litter was produced. At 10000 ppm, one female was found to be not pregnant after positive evidence of mating. A second female at this dose level was found to have a total litter loss in utero. All 1000 ppm females produced a live litter. In the control, one pair failed to mate within the 14 day mating period.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Remarks:

1000 ppm in diet

Sex:

male

Basis for effect level:

other: Local irritation

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Remarks:

1000 ppm in diet

Sex:

female

Basis for effect level:

other: Local irritation

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks:

10000 ppm in diet

Sex:

female

Basis for effect level:

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no inter-group differences in the type or incidence of clinical findings. Findings observed are those commonly seen.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 20000 ppm there was a slightly lower mean litter size at birth compared to control values. The difference was not statistically significant. There was a higher value for the group mean post implantation loss when compared to control values. The large intra-group variability for this parameter means that statistical significance was not achieved. Although group mean litter size was not statistically significantly different to controls, the number of females with litters with 10 or fewer offspring was higher compared to controls (no statistical analysis). Offspring viability between birth and day 4 of lactation was comparable to control values.

At 1000 and 10000 ppm there were no significant effects upon live litter size at birth. Offspring viability between birth and day 4 of lactation was comparable to control values.

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Histopathological findings:

not examined

There were no inter-group differences in the type or incidence of clinical findings. There were no treatment-related effects on the onset or completion of pinna unfolding or the group mean percentages of pups passing the surface righting assessment on Day 1 post partum.

There were no treatment-related macroscopic findings.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 558 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

20 000 ppm in diet

Sex:

female

Basis for effect level:

other: No significant adverse effects

Summary Results

Observations		Dietary Concentration (ppm)
		0 (Control)	1000 ppm	10000 ppm	20000 ppm
Mated pairs (N)	n (number)	9	10	10	10
Females showing evidence of copulation	n	8	10	10	10
Pregnant females	n	8	10	9	10
Conception days 1 -5	n	7	9	10	8
Conception days 6 to 14	n	2	4	1	0
Gestation = 21.5 to 22 days	n	2	4	1	0
Gestation = 22.5 to 23 days	n	5	6	7	6
Gestation = 23.5 to24 days	n	1	0	0	3
Dams with live young born	n	8	10	8	9
Dams with live young at Day 4 post partum	n	8	10	8	10
Corpora lutea/dam	x (mean)	15	18	14	15
Implants/dam	x	14	17	13	14
Live pups/dam at Day 1 post partum	x	14	16	13	11
Live pups/dam at Day 4 post partum	x	13	15	13	11
Sex ratio % males at Day 1 post partum	x	42	53	60	53
Sex ratio % males at Day 4 post partum	x	43	53	60	53
Litter weight at Day 1 post partum	x	92.2	96.7	88.6	74.1
Litter weight at Day 4 post partum	x	118.9	128.0	119.2	109.5
Pup weight at Day 1 post partum	x	6.9	6.1	7.0	6.7
Pup weight at Day 4 post partum	x	9.6	8.3	9.7	10.0
ABNORMAL PUP/DAM
0	n	7	8	6	10
1	n	0	2	2	0
>/= 2	n	1	0	0	0
LOSS OF OFFSPRING/DAM
0	n	2	3	5	5
1	n	4	4	3	2
2	n	1	1	0	1
>/= 3	n	1	1	2	2
Pre-natal (implantation minus live birth)
0	n	5	4	1	2
1	n	2	1	4	1
2	n	1	4	2	3
>/= 3	n	0	0	2	4
Post natal (live births minus offspring alive on Day 4 post partum)
0	n	6	9	8	10
1	n	1	0	1	0
2	n	0	0	1	0
>/= 3	n	1	1	0	0

Conclusions:

The No Observed Effect Level (NOEL) for parental animals was 1000 ppm based on irritancy which was considered an adverse effect. The No Observed Adverse Effect Level (NOAEL) for effects on fertility was 10000 ppm due to increased post implantation loss and consequential decreased mean litter size at birth at the 20000 ppm. The No Observed Effect Level for developmental toxicity/teratogenicity was 20000 ppm.

Executive summary:

This study was designed to screen for potential adverse effect on reproduction including embryo/foetal development in the rat following OECD TG 421 and under GLP conditions.

The test material was administered by dietary admixture to three groups, each of ten male and ten female rats, for up to 54 consecutive days at dietary concentrations of 1000, 10000 and 20000 ppm (equivalent to mean achieved dosages of 64, 651, 1294 and 77, 782 and 1558 mg/kg bw/day for males and females, respectively). A further group of animals served as control. Following 14 days of dosing, male and female rats were paired within their dose groups to produce litters. On Day 5 post partum, all surviving animals were euthanised and examined macroscopically. Clinical signs, bodyweight development and food and water consumption were monitored during the study. Offspring development and growth was also monitored. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and high dose parental males and females was performed.

For adults, there were no treatment-related deaths during the study. At 10000 ppm and 20000 ppm, adverse signs of irritancy were observed in males and females. There were no treatment-related effects on body weight, food consumption or water consumption. Mating performance and fertility were not adversely affected by the treatment. No treatement-related effects were observed for organ weights or necropsy findings.

At 20000 ppm there was in increase in post implantation loss which was not statistically significant compared to controls but was of toxicological significant because of the intragroup variation in individual post implantation loss values. At 10000 ppm there was also a high post implantation loss but this was due to a single female with a total litter loss in utero.

For offspring, at 20000 ppm, there was a slightly lower mean litter size at birth compared to control values but this was not statistically significant. Offspring viability between birth and day 4 of lactation was comparable to control values. For all other observations, there were no treatment-related effects observed.

Administration of the test material to male and female rats throughout maturation, gestation and lactation phrases of reproduction resulted in signs of test material irritancy at 10000 and 20000 ppm. The irritancy was considered to be an adverse effect due to its degree of severity. Therefore, the NOEL for adults was 1000 ppm (66 -74 mg/kg). The NOAEL for effects on fertility was 10000 ppm (782 mg/kg; due to increased post implantation loss and a consequential decrease in mean litter size at birth at 20000 ppm). The NOEL for developmental toxicity/teratogenicity was 20000 ppm (1294 -1558 mg/kg).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

782 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An OECD 421 reproduction/developmental toxicity screening test was conducted in Sprague-Dawley rats by dietary administration at nominal dose levels of 0, 1000, 10000 or 20000 ppm. Toxicologically significant but statistically insignificant post-implantation losses were reported at the two highest dose levels. Signs of irritation were also present at the two highest dose levels. The NOAEL for parental (P1) reproductive effects was set at 782 mg/kg bw/d (10000 ppm, females) level based on increased but statistically insignificant post-implantation losses. The NOEL for parental effects was set at 64 mg/kg bw/d (1000 ppm, males) based on irritaton.

In dietary repeated dose toxicity studies conducted in both rats and dogs, no histopathologically significant effects on reproductive tissues were noted.

Short description of key information:
Results from an OECD 421 reproductive/developmental toxicity screening study in rats

Justification for selection of Effect on fertility via oral route:
A well conducted OECD 421 Guideline study conducted in the rat

Effects on developmental toxicity

Description of key information

The test item was administered daily by oral gavage to pregnant rats from gestation day (GD)-5 up to and including GD20 according to the OECD Test Guideline 414 (June 2018) and following GLP principles.

There was no test item effect on maternal body weight, body weight gain, maternal corrected body weight and body weight gain, and on food intake up to and including 1000 mg/kg bw/day.
There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 1000 mg/kg bw/day.

There were no test item effects on the external, visceral and/or skeletal development of foetuses in the study.

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses. The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL embryotoxicity: 1000 mg/kg bw/day
NOAEL foetotoxicity: 1000 mg/kg bw/day
NOAEL teratogenecity: 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2021-Jan 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 25th 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD®[SD] VAF/Plus®/SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co., Ltd.
- Age at study initiation: 10-11 weeks
- Weight at study initiation: males 315.35-414.25 g; females 190.53-258.47 g
- Fasting period before study: None
- Housing: individually housed in solid bottom cages (43×30×20 cm3 and 34×18×17 cm3) with corncob bedding (except during mating during which the rats were housed on the basis of one male to one or two females)
- Diet (e.g. ad libitum): rodent feed ad libitum; There were no known contaminants (including heavy metals and pesticides) present in the diet expected to interfere with the test results.
- Water (e.g. ad libitum): reverse-osmosis purified and chlorinated water by a water bottle ad libitum; No contaminants were present at levels that would interfere with the outcome of the study.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24.1 °C
- Humidity (%): 36.2-64.4%
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 2021-01-08 To: 2021-02-05

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% (w/v) CMC-Na in purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared under yellow light. The required amount of test item was added ton 70% of the final volume of vehicle while stirring. The formulation was stirred until a homogenous formulation was formed by visual inspection. The formation was brought to its final volume. The formulations intended for dosing were stirred at room temperature for more than 30 minutes before dosing and continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water. Previous experimental studies have shown that dose preparations are adequately prepared using 1% CMC.
- Concentration in vehicle: 1% in purified water
- Amount of vehicle (if gavage): 10 ml
- Lot/batch no. (if required): c2030035

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate samples (one for analysis, the other two for backup) were collected from the middle layer of the mid-dose formulations and control formulations for the first and last preparations. For the low- and high-dose formulations, mean results of the homogeneity results were used as concentration verification and no additional samples were collected.

For homogeneity analysis, triplicate samples (one for analysis, the other two for backup) were collected from the low and high-dose formulations from the top, middle, and bottom layer of formulations for the first and last preparations.

The concentration and homogeneity results of the test item in the dosing formulations met the acceptance criteria, which demonstrated the formulations were accurately prepared and homogenous.
The concentrations of the test item in the dosing formulations were within 101% to 105% of nominal values.
There was no detectable peak observed at the retention time of the test item when analyzing control samples. The result was reported as “Homogeneity analysis of the low- and high-dose dosing formulations was performed. The concentrations of the top, middle and bottom of samples were within 100% to 106% of nominal values. The relative standard deviations (RSD) of top, middle, and bottom of samples were within 0.4% to 3%.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male to one or two female(s)
- Length of cohabitation: Until copulation has occurred, max 3d
- After 3 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not applicable
- Verification of same strain and source of both sexes: Both sexes were the same strain and obtained from the same supplier at a single time.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- Any deviations from standard protocol: No

Duration of treatment / exposure:

Oral administration from Gestation Day 5 to 20 (included)

Frequency of treatment:

Once daily

Duration of test:

From Gestation Day 5 to 20

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Remarks:

Low-dose

Dose / conc.:

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

Remarks:

Mid-dose

Dose / conc.:

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

Remarks:

High-dose

No. of animals per sex per dose:

25 mated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosages were selected based on the preliminary dietary exposure study in rats (OECD421). In that study, the NOEL for developmental toxicity was 1400 mg/kg (highest dose tested). Due to post implantation loss and decrease in mean litter size at birth, the NOEL for fertility was 780 mg/kg. In a 90-day repeated dose toxicity study with rats (dietary exposure, no guideline as this predates the guidelines) the NOAEL was 400 mg/kg (highest dose tested).
In addition, a 7-day tolerability study was conducted prior this study (January 2021) to ensure that administration by gavage compared to dietary exposure in the studies listed above would not lead to unexpected toxicity.
Based on all study results, 1000 mg/kg/day was selected as the highest dose level for this study (maximum dose recommended in the OECD TG 414), and 100 and 300 mg/kg were selected as low and mid doses, respectively.

- Rationale for animal assignment (if not random): Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during gestation except for days on which detailed observations are conducted; Unscheduled observations will be made when a change in condition or behavior is noted.
- Cage side observations: See details of observations in the tables included as attached background material.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before assignment, Once daily on GD0, and from GD5 to GD21.

BODY WEIGHT: Yes
- Time schedule for examinations: Once prior cohabitation, at least once on GD 0, 3, 5, 6, 9, 12, 15, 18, 20, and 21.
The corrected body weight calculated on the day of necropsy (body weight on GD21 minus weight of the gravid uterus).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Brain, Thyroid glands with parathyroid gland(s), Gross lesions (See details of observations in the tables included as attached background material).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No sampling, gross evaluation performed
- Serum: Yes
- Volume collected: 3 mL

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (control and high-dose)
- Skeletal examinations: Yes: half per litter (control and high-dose)
- Anogenital distance of all live rodent pups: Yes
- Sex, body weight, crown-rump length of all live rodent pups: Yes

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea-No. of implantations)/No. of corpora lutea ×100%

Post-implantation loss was calculated as a percentage from the formula:
(No. of implantations-No. of live fetuses)/No. of implantations ×100%

Sex ratios of the live fetuses were calculated as the percentage of males per litter.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical signs observed, including alopecia, sore, and material in bedding (yellow food-like), were considered incidental due to low incidence and/or similar findings in the control animals, and therefore considered not to be test item-related.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy

Body weight and weight changes:

no effects observed

Description (incidence and severity):

All variations in body weight were small in magnitude or comparable with the concurrent control group. Therefore, they were considered unrelated to test item treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

All variations in food consumption were small in magnitude or comparable with the concurrent control group. Therefore, they were considered unrelated to test item treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

No statistically significant changes were observed for TSH, T3 or T4 in this study.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related organ weight changes at scheduled termination.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic changes in thyroid glands with parathyroid gland (s) at 1000 mg/kg/day. All microscopic findings present in this study were considered background or incidental changes of rats of this age.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

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

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

One of 346 fetuses at 100 mg/kg/day was observed with general subcutaneous edema malformation. This external malformation was considered a spontaneous finding and not test item-related due to low incidence and being within the range of historical control data in this laboratory.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The skeletal variations, including short rib, wavy rib, interrupted costal cartilage, unossified hyoid, incomplete ossification of sternebra, unossified sternebra, short supernumerary rib, and dumbbell-shaped and bipartite ossification of thoracic centrum were observed in treated and/or control groups. These findings were considered not test item-related due to similar findings occurring in the treatment and control groups and/or because of the low incidence (the incidence was within the range of historical control data) in this laboratory.

One of 175 fetuses at 1000 mg/kg/day was observed with costal cartilage fused malformation (the left 10th and 11th fused). This malformation was considered a spontaneous finding and not test item-related due to low incidence.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One of 165 fetuses at 1000 mg/kg/day was observed with situs inversus malformation. This visceral malformation was considered a spontaneous finding and not test item-related due to the low incidence.
The visceral variations including dilated renal pelvis, convoluted ureter, and dilated ureter were observed at treated group and/or at control, and were considered not test item-related due to similar findings being present in the control, and/or low incidence (the incidence was within the range of historical control data in this laboratory).

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

See tables available in the attached background document.

Conclusions:

The test item was administered daily by oral gavage to pregnant rats from gestation day (GD)-5 up to and including GD20 according to the OECD Test Guideline 414 (June 2018) and following GLP principles.

There was no test item effect on maternal body weight, body weight gain, maternal corrected body weight and body weight gain, and on food intake up to and including 1000 mg/kg bw/day.
There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 1000 mg/kg bw/day.

There were no test item effects on the external, visceral and/or skeletal development of foetuses in the study.

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses. The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL embryotoxicity: 1000 mg/kg bw/day
NOAEL foetotoxicity: 1000 mg/kg bw/day
NOAEL teratogenecity: 1000 mg/kg bw/day

Executive summary:

The purpose of this study was to investigate the effects of the test item on embryo-fetal development in rats when administered orally from Gestation Day (GD) 5 up to and including GD 20 and to characterize the dose-response relationship of any observed toxicity according to the OECD TG 414 and following GLP. 

One hundred (100) mated female rats were randomly assigned to 4 groups with 25 animals in the control and treatment groups.  Animals were administered the test item at dose levels of 0 (control formulation, 1% (w/v) CMC-Na in purified water), 100, 300, and 1000 mg/kg/day by oral gavage once daily from GD 5 to 20. The dose volume was 10 mL/kg. At initiation of mating, male and female rats were approximately 10 to 11 weeks of age and the male animal body weights were 315.35 to 414.25 g and the female animals were 190.53 to 258.47 g.  Females were nulliparous and non-pregnant before cohabitation.

The study animals were observed daily for mortality and clinical signs, and measured for body weight and food consumption during this study.  On GD 21, all study animals were necropsied and examined for gross pathology, organ weights, and histopathology (control and high dose groups).  Blood samples were collected to conduct thyroid stimulating hormone (TSH) and thyroid hormones T3 and T4 analysis from all study animals at the scheduled necropsy. The ovaries and uteri were examined for determination of litter data. Uteri without visible implantation were placed in ammonium sulfide solution for detection of early resorptions. The placenta of live fetuses was examined macroscopically. All the live fetuses were weighed and examined for external abnormalities; their crown-lump lengths were measured and sexes were determined. Approximately 1/2 of the live fetuses in each litter were fixed with modified Davidson’s fixative for soft tissue examination and the remaining fetuses were stained with Alcian Blue Solution and Alizarin Red Solution for subsequent skeletal examination. The visceral and skeletal examination of fetuses was conducted for the high dose and control groups.

The concentrations of the test item in dosing formulations (which were used to dose) were within 101% to 105% of nominal values. Homogeneity analysis of the low and high-dose formulations was performed. The concentrations of the top, middle and bottom of samples (in dosing formulation which were used to dose) were within 100% to 106% of nominal values. The relative standard deviations (RSD) of top, middle, and bottom of samples were within 0.4% to 3%.

All animals survived to the scheduled necropsy. There were 23/25, 25/25, 19/25, and 23/25 pregnant animals in 0, 100, 300, and 1000 mg/kg/day, respectively.

There were no test item-related changes in the clinical signs, body weights, body weight changes, gravid uterine weight, food consumption, gross pathology, organ weights, and histopathology of the dams.

There were no test item-related changes in litter data (number of corpora lutea, implantation sites, live fetuses, fetal death, dead fetuses, resorptions, pre‑ and post- implantation loss, fetal weight and fetal crown-rump length), sex ratio, placental examination, the anogenital distance or fetal structure (visceral and skeletal examinations).

There was no test item-related changes observed in this study for thryoid hormones (T3, T4, and TSH).

In conclusion, administration of the test item to pregnant female rats once daily during the period of organogenesis (from Gestation Day 5 to 20) by oral gavage at dosages of 0, 100, 300, and 1000 mg/kg/day was well tolerated and did not result in any treatment-related mortality, and any adverse effects in clinical signs, body weight, food consumption, and pathology.  No test item-related embryo-fetal developmental toxicity was noted at any dose level.  The No-Observed-Adverse-Effect-Level (NOAEL) for all was considered to be 1000 mg/kg/day (the highest dose tested).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

There were no significant developmental or teratogenic effects noted in offspring in an OECD 414 study. The NOAEL for developmental effects in this study was reported as 1000 mg/kg bw/day, the highest dose level tested.

Justification for selection of Effect on developmental toxicity: via oral route:
A well conducted OECD 414 Guideline study conducted in the rat

Justification for classification or non-classification

Minimal but statistically insignificant reproductive effects were noted in an OECD 421 study in rats as post-implantation losses. These effects were not seen in the OECD 414 conducted subsequently. No significant effects were reported in reproductive tissues from chronic toxicity testing in either rats or dogs. Based on these findings, no classification of the test material for reproductive effects is warranted.

Based on negative finding for developmental effects in an OECD 414 study in rats, no classification of the test material for developmental toxicity is warranted.