Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

There were no adverse effects on fertility observed.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 Feb 2018 to 23 May 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
yes
Remarks:
Clinical chemistry including hormone determinations were not performed
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: 0000091655
- Expiration date of the lot/batch: 31 August 2018, The test item expired during the experimental phase of the study as per the declared expiry date on the CoA (Certificate of Analysis). To establish the stability of the test item, the active ingredient content will be analysed concurrently with the dosing period i.e., within one week before declared expiry and within one week after completion of dosing following the validated analytical method. The CoA and comparison of analysed purity were reported in the final report.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As per the instruction received from the Sponsor on storage of the test item, the test item will be stored:
- Storage Temperature: Room Temperature
- Storage Container: In original container as supplied by the Sponsor
- Stability under test conditions: The test item is stable up to 14 days at room temperature based on the results of the method validation study. The test diet will be prepared at least once every 8 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The required quantity of the test item was received from the Test Item Control Office (TICO). The test item was mixed with the 10% of total diet or 500 g of food (whichever is higher) in order to prepare the pre-mix. To this pre-mix diet, the remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in a blender and served to the animals ad libitum throughout the study. Details of the diet preparation were recorded in the raw data.
Species:
rat
Strain:
Wistar
Remarks:
RccHan
Details on species / strain selection:
Rat is the preferred test system because it is a readily available laboratory animal and is historically shown as acceptable animal for the repeated dose toxicity studies and recommended by the OECD and other regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 wks
- Range of mean body weight per group on Day 1: (P) Males: 368.59 - 371.29 g; Females: 235.54-237.14 g. At initiation of treatment, the body weight of rats (P) was within ±20% of the mean body weight for each sex.
- Range of mean body weight per group on PND0: (F1) Males: 5.16-5.53 g; Females: 4.82-5.24 g
- Housing: Throughout the experiment period, the male and female rats were housed individually and enrichment material were provided. During the mating period, rats were housed in groups of 2 rats/cage (one male plus one female). Mated female rat were caged individually and nesting material was provided at near parturition. During study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless steel top grill having provision for polypropylene water bottle with stainless steel drinking nozzle. Separate feed hoppers were attached to the cages for diet. The bottom of cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on 5/6 tier racks. Cages were changed at least twice a week. Cages were arranged in a such way that possible effects due to cage placement are minimised. Contaminant analysis of samples of the bedding material is being performed half yearly intervals.
- Diet: ad libitum with standard rodent diet.
- Water: unlimited supply of clean and filtered drinking water in polypropylene bottles.
- Acclimation period: A total of 36 male and 36 female rats were selected for acclimatisation, after due consideration. Rats were received into the experimental room and acclimatised for a minimum period of 5 days prior to oestrous cycle evaluation. During acclimatisation period, rats were observed daily, at least once, for clinical signs.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATE:
27 Feb 2018 to 23 May 2018
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was mixed with the 10% of total diet or 500 g of food (whichever is higher from the total quantity of total diet) in order to prepare the pre-mix. To this pre-mix diet, remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in blender and served to animals ad libitum throughout the study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurs or 2 weeks have elapsed
- Proof of pregnancy: ‘Day 0’ of pregnancy was defined as the day on which sperms were observed in the vaginal smears of rats.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually and nesting material was provided at near parturition.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The active ingredient concentration and homogeneity of the test item in diet was analysed once before initiation of treatment and twice during treatment period. Triplicate samples from different locations (i.e., top, middle, and bottom of container) of the test diet from each group was taken to determine the homogeneity and concentration of the test item in the test diets. Duplicate samples were collected and one set was sent for analysis and second set were stored at 2-8 °C. Stored samples were used, if re-analysis was required. Every time, the mean concentration will be determined and compared with the nominal value. During the study, the mean percent recovery (MR) and coefficient of variation (CV) obtained for different test diet concentrations given to rats during the treatment period at different intervals are mentioned in Table 1 in "Any other information on results, incl. tables".
Duration of treatment / exposure:
During the treatment period, rats were offered the control or test diets continuously on a 7-days/week basis, till necropsy. Exposure of rats, belonging to both sexes, were initiated at least 2 weeks prior to mating and continued during the mating period. After mating, male rats were further exposed up to and including the day before scheduled sacrifice (until approximately 80% of the females have delivered). Female rats were further exposed during gestation and at least up to post-partum day 13.
Frequency of treatment:
Continuous
Dose / conc.:
150 ppm
Remarks:
Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively
Dose / conc.:
300 ppm
Remarks:
Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively
Dose / conc.:
600 ppm
Remarks:
Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
8
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on the results of dose range finding study.
- Other: On the postnatal day four, the size of each F1 litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four male and four female pups per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (e.g., six males and two females) was performed. If possible, the two pups/litter were female pups and male pups were reserved for nipple retention observation. Adjustments were not done for litters of eight pups or less.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily, at least twice for mortality and morbidity and once for clinical signs
- Observations: mortality and morbidity. Rats, which died or sacrificed during moribund condition in the course of study, were weighed and subjected to post-mortem examination. Female rats, which show marked signs of reaction to treatment or premature delivery, were sacrificed and examined.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, at least once, throughout the study
-Clinical observations: Visible clinical signs, such as changes in skin, fur, eye, mucous membranes as well as behaviour pattern and abortion.

BODY WEIGHT:
- Time schedule for examinations: Body weight of all male animals was recorded on the first day of treatment and at weekly intervals thereafter. Body weight of all female animals was recorded on the first day of treatment and at weekly intervals during pre-mating and mating periods. During the gestation period, female rats were weighed on GDs 0, 7, 14, and 20. During the lactation period, female animals were weighed within 24 hours of parturition (day '0' post-partum/lactation day), and on post-partum days 4, 7, 14 and on day of terminal sacrifice. Parturition day '0' is defined as the day on which the female littered. On day of fasting, body weights of all surviving rats were recorded. Body weights of all the rats were also recorded on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as food input (g) - Food leftover (g)
- The food consumption was determined by differentiating the weight of feed input and leftover. Food weights of male rats were measured weekly, at least once, during pre-mating and postmating period. In female rats, during pre-mating period food weights were recorded weekly, at least once. During gestation period, food weights were measured at least on days 0, 7, 14, and 20. During lactation period, food weights were measured at least on lactation days 0, 4, 7, 14 and on day of terminal sacrifice. Additional food were offered as and when required. Food consumption was not measured during mating period. Food spillage/waste may not have been estimated during the period. However, evidence of excessive spillage was recorded in the raw data.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data
Oestrous cyclicity (parental animals):
Vaginal smear were monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant animal, were also observed on the day of terminal sacrifice. Care was taken to avoid disturbance of mucosa while obtaining vaginal cells.
Litter observations:
Each litter was examined after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Ano-genital distance (AGD) of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13. Individual pup body weight was recorded at least on lactation days 0, 4, 7, and 14.
Postmortem examinations (parental animals):
- Surviving rats were sacrificed by using an over dose of carbon dioxide. Gross necropsy was conducted under direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, open, and a thorough examination of organs were carried out to detect abnormalities. Special attention was paid to organs of the reproductive system. Gross examination of animals were conducted. Male rats were sacrificed after approximately 80% of females have delivered. Female rats were sacrificed on post-partum day 15. Females which did not mate were sacrificed during 24-26 days after the last day of mating period. Females which have not delivered by day 25 post-coitum were sacrificed on post-coitum day 25.
- At the time of sacrifice or death during the study, adult rats were examined macroscopically for any structural abnormalities or pathological changes. Particular attention were paid to the external reproductive genitals which were examined for signs of altered development. Number of implantation sites were recorded for each dam (including rats subjected to moribund sacrifice or found dead). Special attention was given to the organs of the reproductive system.
- The liver, kidney, brain, and spleen were weighed from the surviving parental rats. The testes, epididymis, prostate and seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper’s glands, and glans penis of all parental male and paired ovaries (wet weight) and uterus (including cervix) in females rats were weighed. The thyroid with parathyroid weight were determined after fixation from all surviving parental rats.
Postmortem examinations (offspring):
Surviving rats including pups of PND 14 were humanely sacrificed by using an over dose of carbon dioxide. Culled pups on PND 4 were sacrificed through intraperitoneal injection of thiopentone sodium. Pups which died during lactation were weighed and subjected to post-mortem examination.
Gross necropsy was conducted under direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities. Special attention was paid to organs of the reproductive system. Gross examination of pups was conducted. At the time of sacrifice, pups were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Pups were sacrificed on post-partum day 14. Particular attention were paid to the external reproductive genitals which were examined for signs of altered development. Special attention was given to the organs of the reproductive system. Thyroid with parathyroid from 1 male and 1 female pup per litter per group were weighed after fixation.
Statistics:
The statistical analysis was carried out for the parameters using validated software developed at JRF. Non-pregnant animals were excluded from statistical analysis. Data such as body weight, body weight gain, food consumption, food efficiency, litter size, organ weight, organ weight ratio, number of implantation, pre-natal loss, post-natal loss, and litter parameters [male pups, female pups, total pups (male + female) counts and pups weight] were analysed using Bartlett's test of homogeneity of variance. When the result was not significant then an analysis of variance (ANOVA) was carried out. When the ANOVA was significant then a Dunnett's multiple comparison test was carried out. When the data did not meet the homogeneity of variance then a t-test was performed to calculate significance at 95 and 99% confidence levels. AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis. Non-parametric data such as mortality rate, gestation index, parturition index, pregnancy rate, pup survival index, live birth index and fertility index were analysed using a Chi-Square test. Flags for significant difference between control and treated groups (single arrow for p<0.05 and double arrows for p<0.01) were given in the tables along with the footnote.
Reproductive indices:
In detail all indices are displayed in the field 'Any other information on materials and methods, inlc. tables'.
Offspring viability indices:
In detail all indices are displayed in the field 'Any other information on materials and methods, inlc. tables'.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight gain of male rats, belonging to the 600 ppm dose group, was statistically significant decreased during treatment days 1-8, 29-36, and 1-43, when compared with that of the control group. The mean body weight gain of female rats, belonging to the 600 ppm dose group, was also statistically significant decreased during pre-mating days 1-8 and 1-15, when compared with that of the control group. The mean body weight gain of female rats, belonging to the 600 ppm dose group, was also statistically significant decreased during Lactation Day 4-7 and 0-14, when compared with that of the control group. The mean body weight of female rats, belonging to the 600 ppm dose group, was statistically significant decreased on Lactation Day 14, when compared with that of the control group. These decreases in mean body weight and body weight gain could be due to a decrease in mean food consumption of rats belonging to the 600 ppm dose groups. These effects could be considered as treatment related adverse effects of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- The mean food consumption of male rats, belonging to the 600 ppm dose group, were statistically significant decreased during the treatment days 1-8, 28-36, 1-43, and 1-8, 1-43, respectively, when compared with that of the control group. The mean food consumption of male rats, belonging to the 300 ppm dose group, were statistically significant decreased during the treatment days 28-36, when compared with that of the control group. The mean food consumption of male rats, belonging to the 150 ppm dose group, were statistically significant decreased during the treatment days 1-8, 28-36, and 1-43, when compared with that of the control group. The mean food consumption of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the premating days 1-8, 8-15, 1-15, and 1-8 and 1-15, respectively, when compared with that of the control group. The mean food consumption of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the Lactation Days 4-7 and 0-14, and 4-7, 7-14, 0-14, respectively, when compared with that of the control group. The mean food consumption of female rats, belonging to the 300 ppm dose group, were statistically significant decreased during the Lactation Days 4-7, when compared with that of the control group. The mean food consumption of female rats, belonging to the 150 ppm dose group, were statistically significant decreased during the pre-mating days 1-8, when compared with that of the control group. These decreases in mean food consumption of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. An occasional decrease in mean food consumption without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance.
- The overall mean test item intake of male rats, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 8.59, 17.59, 33.53 mg/kg b. wt./day, respectively. The overall mean test item intake of female rats during pre-mating, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 10.86, 22.46, 39.85 mg/kg b. wt./day, respectively. The overall mean test item intake of female rats during gestation, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 10.47, 20.96, 42.19 mg/kg b. wt./day, respectively. The overall mean test item intake of female rats during lactation, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 18.78, 33.92, 66.91 mg/kg b. wt./day, respectively.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The mean food efficiency of male rats, belonging to the 600 ppm dose group, were statistically significant decreased during the treatment days 1-8, 28-36, 1-43, and 1-8, 1-43, respectively, when compared with that of the control group. The mean food efficiency of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the premating days 1-8, 8-15, 1-15, and 1-8 and 1-15, respectively, when compared with that of the control group. The mean food efficiency of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the Lactation Days 4-7 and 0-14, and 4-7, 7-14, 0-14, respectively, when compared with that of the control group. These decreases in mean food efficiency of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. An occasional decrease in mean food efficiency without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in mean absolute and relative liver weights were observed in male rats, belonging to the 600 ppm dose group. This effect was also observed in male rats of the 300 ppm dose group with less severity. These increases in mean absolute and relative liver weights of male rats, belonging to the 300 and 600 ppm dose groups, were considered as a treatment related effect of the test item.
A statistically significant decrease was observed in mean terminal body weight of female rats, belonging to the 600 ppm dose group. The marginal decrease was also observed in mean terminal body weight of male rats, belonging to the 600 ppm dose group. The effect could be related to test item treatment. Results presented in Table 2 in "Any other information on results, incl. tables".
Statistically significant increases were observed in mean relative weights of the brain in male and female rats, belonging to the 600 ppm dose group. The effect was considered as an indirect effect due to the decrease in body weights.
A statistically significant decrease was observed in mean absolute weight of the spleen of female rats, belonging to the 600 ppm dose group. However, a statistically significant increase was observed in mean relative weight of kidneys of male rats, belonging to the 600 ppm dose group. These effects were not considered treatment related due to the absence of consistency between sexes and consistency between absolute and relative weights. Similarly, a statistically significant decrease was observed in mean absolute weight of the levator ani plus bulbocavemosus muscle complex (LABC) of male rats, belonging to the 300 ppm dose group. It was not considered as treatment related due to the absence of dose dependency.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
External examination of terminally sacrificed male and female rats, belonging to 0, 150, 300, and 600 ppm dose groups did not reveal any abnormality.
Greyish discolouration of liver of male (8/8) and female (8/8) rats, belonging to the 600 ppm dose group, was observed during necropsy. Greyish discolouration of liver of male (3/5) and female (7/8) rats, belonging to the 300 ppm dose group, was also observed during necropsy. These alterations were considered as treatment related effects of the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Mean estrous cycle length and pattern of estrous cycle were comparable with the control group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related differences were observed in sex ratio and mean counts of male pups, female pups, and pups of both sexes combined when compared with that of the control group.
The live birth index of female pups and pups of both sexes combined, belonging to the 300 ppm dose group, was statistically significant decreased, when compared with that of the control group. However, no dose dependency was observed. Therefore this could be considered incidental without toxicological relevance.
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other:
Remarks:
Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
> 600 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
All pups belonging to control and test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mortality of male pups, belonging to the 600 ppm dose group, were statistically significant increased during the PNDs 0-4, when compared with that of the control group. The mortality of male pups and pups of both sexes combined, belonging to the 300 ppm dose group, were statistically significant increased during the PNDs 0-4, when compared with that of the control group. The mortality without significant effect on body weight (PNDs 0 and 4) and body weight gain (PNDs 0-4) of pups, belonging to the 600 ppm dose group, could be considered as an incidental finding without any toxicological relevance. The mortality of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised, and was not considered as treatment related.
The survival index of male pups, belonging to the 600 ppm dose group, were statistically significant decreased on PND 4, when compared with that of the control group. The survival index of male pups and pups of both sexes combined, belonging to the 300 ppm dose group, were statistically significant decreased on PND 4, when compared with that of the control group. The decrease in survival index without significant effect on body weight and body weight gain of pups, belonging to the 600 ppm dose group, could be considered as incidental without any toxicological relevance. The decreased survival index of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised, and was not considered as treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of male pups, female pups, and pups of both sexes combined, belonging to the 600 ppm dose group, was statistically significant decreased on PND 14, when compared with that of the control group. The mean body weight gain of male pups, female pups, and pups of both sexes combined, belonging to the 600 ppm dose group, was statistically significant decreased on PNDs 7-14, when compared with that of the control group. The mean body weight gain of female pups and pups of both sexes combined, belonging to the 600 ppm dose group, was statistically significant decreased on PNDs 4R-7, when compared with that of the control group. These decreases in mean pups body weight and body weight gain could be due to a decrease in mean food consumption and food efficiency of female rats of the 600 ppm dose group which subsequently might not produce sufficient milk for the pups. This effect could be considered as a treatment related adverse effect of the test item on pups growth.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance measured on PND 0 of the test item treated groups was comparable with that of the control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
None of the male pups belonging to either control or the test item treatment groups showed retention of nipples.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease was observed in mean terminal body weight of male and females pups, belonging to the 600 ppm dose group. The effect was considered as a treatment related effect of the test item. A statistically significant increase in relative weights of the thyroid with parathyroid was observed in male and female pups, belonging to the 600 ppm dose group. This effect without statistical significance was also observed in absolute weights and was considered as a treatement related effect of the test item. See results in Table 3 in "Any other information on results, incl. tables'.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
External and internal examination of pups (found dead and terminally sacrificed on PNDs 4 and 14) did not reveal any abnormality. One found dead pup was autolysed on internal examination and the milk band was absent in all the found dead pups.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
Developmental Toxicity
Generation:
F1
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other:
Remarks:
Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively
Critical effects observed:
yes
Lowest effective dose / conc.:
600 ppm
System:
endocrine system
Organ:
thyroid gland
parathyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Reproductive effects observed:
no

Table 1. Homogeneity and Active Ingredient Concentration Analysis of the Test Item in the Diet

Group N°

G1

G2

G3

G4

Dose (ppm)

0

150

300

600

Interval

MR
CA)

% CV

MR (%)

% CV

MR (%)

% CV

MR (%)

% CV

Before treatment

-

-

99.98

2.95

96.68

4.10

105.54

0.15

During treatment

-

-

84.09

1.56

97.84

1.93

88.18

1.89

During treatment

-

-

85.61

4.15

81.20

0.67

96.00

0.07

 Key: - = Not applicable, MR = Mean recovery, % = Percent, CV = Coefficient of variation

The mean percent recovery obtained for the test diet was within the acceptance level of ±20% of the nominal concentration demonstrating that the exposure concentrations were as intended by the study plan and the %CV was less than 20, suggesting that the test diet were homogeneously mixed.

Table 2. Liver weight result.

Sex

Liver

Groups

% increase/decrease
than control

G1

G2

G3

G4

G2

G3

G4

Male

Absolute weight

(g) SD)

(Mean ±

12.090 ±

1.622

12.493 ±

0.653

13.958 ±

0.88411'

14.840 ±

0.93211'

3

15

23

Relative weight (g) (Mean ± SD)

2.862 ±

0.297

2.991 ±

0.141

3.335 ±

0.23311'

3.683 ±

0.26711'

5

17

29

 Key: 1r= significantly higher than control (1)0.01)

PND 14 Pups:

Table 3: Terminal body weights and thyroid with parathyroid (relative and absolute)

Sex

Organ

Groups (Mean ± SD)

% increase/ decrease than control

G1

G2

G3

G4

G2

G3

G4

 

 

24.800 ±

23.375 ±

22.383 ±

18.283 ±

 

 

 

 

B. wt. (TS)

 

 

 

 

-6

-10

-26

 

 

1.698

4.335

4.868

2.282 J.

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0283 ±

0.0295 ±

0.0319 ±

0.0453 ±

 

 

 

 

parathyroid-

 

 

 

 

4

13

60

M

relative

0.0036

0.0129

0.0095

0.0118T

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0070 ±

0.0066 ±

0.0071 ±

0.0082 ±

 

 

 

 

parathyroid-

 

 

 

 

-6

1

17

 

absolute

0.0009

0.0022

0.0023

0.0016

 

 

 

 

 

23.667 ±

23.563 ±

21.200 ±

18.843 ±

 

 

 

 

B. wt. (TS)

 

 

 

 

0

-10

-20

 

 

2.884

2.170

3.278

3.637

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0276 ±

0.0284 ±

0.0276 ±

0.0481 ±

 

 

 

 

parathyroid-

 

 

 

 

3

0

74

F

relative

0.0069

0.0072

0.0068

0.0153 T T

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0065 ±

0.0066 ±

0.0058 ±

0.0087 ±

 

 

 

 

parathyroid-

 

 

 

 

2

-11

34

 

absolute

0.0017

0.0013

0.0013

0.0020

 

 

 

 

Keys: M = Male, F = Female, T = significantly higher than control (p0.05), J. = significantly lower than control (p0.05) T T= significantly higher than control (p0.01)

A statistically significant decrease in terminal body weights (20 to 26 % decrease compared to control) was noted in male and female pups of the high dose group. The effect was related to treatment of the test item.

A statistically significant increase in relative weights of thyroid with parathyroid (60 to 74 % increase compared to control) was noted in pups of the high dose group of both sexes. The effect without statistical significance was also noted in absolute weights (17 to 34% increase compared to control) and was considered as related to test item treatment.

Conclusions:
- Based on the results of the present study, the following dose levels are suggested for the extended one-generation reproductive toxicity study (cohort-1) of the test item in Wistar rats: 75, 150, and 300 ppm.
- NOAEL systemic toxicity: 150 ppm, based on the gross pathology abd greyish discoloration of the liver, both sexes (Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively)
- NOAEL reproductive toxicity: >600ppm, based on no adverse toxic effect observed, both sexes (Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively)
- NOAEL developmental toxicity: 300 ppm, based on the effect on the thyroid and parathyroid gland weight and the body weight ratios, both sexes (Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively)
Executive summary:

The study was conducted under GLP conditions to determine the initial information on toxic characteristic, systemic and reproduction/developmental toxicity, occurring as a result of repeated daily dietary administration of the test item during pre-mating, mating, gestation, and lactation period of rats to select the dose levels for definitive study. A total of 36 male and 36 female Wistar rats were randomly divided into four groups (8 rats/sex/group). Test item was given through diet, in graduated doses of 150, 300, and 600 ppm, to three groups of male and female rats. The control group received basal diet. Male rats were treated for 43 days. Female rats were treated for two weeks prior to mating, variable time to conception, the duration of pregnancy and 14 days after delivery. The active ingredient concentration and homogeneity of the test item in diet were analysed once before the initiation of treatment and twice during the treatment period. Oestrous cycle length and pattern of female rats were evaluated by vaginal smears during pre-treatment of two weeks and daily from the beginning of the treatment period until evidence of mating. Rats were observed daily, twice, for mortality and morbidity, and once for visible clinical signs throughout the study period. Body weight of rats was recorded on the first day of treatment and the day of sacrifice. Body weight of male rats were recorded weekly throughout the treatment period. Body weights of female rats were recorded weekly during pre-mating and mating periods. Pregnant females were weighed on gestation days (GDs) 0, 7, 14, 20, within 24 hours of parturition, and post-coitum on day 25. Body weights of dams and pups were recorded on lactation days 0, 4, 7, and 14. Food consumption and test item intake was calculated during the treatment period. Pups were observed for sex, stillbirths, live birth, runts, gross anomalies, and measured for ano-genital distance (AGD) on post-natal day (PND) 0. Each litter was standardised on PND 4 to obtain nearly 4 male and 4 female pups. Rats were terminally sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination. Absolute organ weights were recorded and relative organ weights were calculated.

The results of diet formulation analyses were within the acceptable range of ±20% of the nominal concentration and the %CV was less than 20 which suggests that the prepared diet had an acceptable a.i. concentration and it was homogeneously prepared. Parents: No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 600 ppm dose level. The mean body weight gain, food consumption, and food efficiency of male rats, belonging to the 600 ppm dose group, was statistically significant decreased during treatment, when compared with that of the control group. The mean terminal body weight of female rats, belonging to the 600 ppm dose group, was statistically significant decreased, when compared with that of the control group. The mean body weight gain, food consumption, and food efficiency of female rats, belonging to the 600 ppm dose group, was statistically significant decreased during the pre-mating and lactation period, when compared with that of the control group. These decreases in mean body weight gain, food consumption, and food efficiency of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. Occational decreases in mean food consumption and food efficiency without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance. External examination of terminally sacrificed male and female rats, belonging to the 0, 150, 300, and 600 ppm dose groups did not reveal any abnormality. Greyish discolouration of liver of male and female rats, belonging to the 300 and 600 ppm dose groups, was observed during necropsy. Treatment related increases in mean absolute and relative liver weights of male rats, belonging to the 300 and 600 ppm dose groups was observed. Pups: Pup mortality was observed in litters, belonging to the 300 and 600 ppm dose groups. However, the body weight and body weight gain of pups, belonging to the 600 ppm dose group was comparable. Therefore, this could be considered as an incidental change. The mortality of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised and was not considered as treatment related. Pups mortality was also reflected in live birth and survival indices. The mean body weight and body weight gain of pups, belonging to the 600 ppm dose group, was statistically significant decreased during PNDs 7-14, when compared with that of the control group. This could be due to a decrease in mean food consumption and food efficiency of female rats of the 600 ppm dose group which might not produce sufficient milk for the pups. No treatment related differences were observed in litter size, sex ratio, AGD, and nipple retention of the pups. External and internal examination of the pups did not reveal any abnormality. A statistically significant increase in relative weights of thyroid with parathyroid was observed in male and female pups, belonging to the 600 ppm dose group.

Based on the results of the present study, the following dose levels are suggested for the extended one-generation reproductive toxicity study (cohort-1) of the test item in Wistar rats: 75, 150, and 300 ppm. The following NOAELs were determined based on the study:

- NOAEL systemic toxicity: 150 ppm, based on the gross pathology abd greyish discoloration of the liver, both sexes (Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively)

- NOAEL reproductive toxicity: >600ppm, based on no adverse toxic effect observed, both sexes (Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively)

- NOAEL developmental toxicity: 300 ppm, based on the effect on the thyroid and parathyroid gland weight and the body weight ratios,  both sexes (Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively)

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
OECD 421. Under GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The objective of this study was to evaluate the prenatal developmental toxicity of the test material when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits.This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. Mated female rabbits were assigned to four groups with 23 animals in each group. The day on which mating had occurred was considered as gestation Day 0 (GD 0) for each individual female rabbit. Test material in vehicle (Corn oil) was administered at 0, 5, 15 and 50 mg/kg/day. The control group received the vehicle only. Dose formulation analysison initiation and termination of treatment periodindicated all samples were within 15% of the nominal concentrations. All rabbits were observed for clinical signs, morbidity and mortality, body weight changes and food consumption.Caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses.The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. The main findings of the study are presented below:

There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested except for incidental abortions in each of the, low, mid and high dose groups. Mean body weight gain at 50 mg/kg/day was lower compared to the vehicle control animals. Food consumption at 50 mg/kg/day was reduced during treatment period (GD 6 to 28). At 50 mg/kg/day there was a reduction in uterine weight, increase in post implantation loss and two dams with complete resorptions. At 50 mg/kg/day a significant reduction in litter weight was observed. Visceral evaluation at 50 mg/kg/day showed incidences (2/94 fetus) of unilateral absence of kidney and ureter and ureter dilated with hydronephrosis.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) for maternal and fetal developmental toxicity is 15 mg/kg/day.

The study was conducted under GLP conditions to determine the initial information on toxic characteristic, systemic and reproduction/developmental toxicity, occurring as a result of repeated daily dietary administration of the test item during pre-mating, mating, gestation, and lactation period of rats to select the dose levels for definitive study. A total of 36 male and 36 female Wistar rats were randomly divided into four groups (8 rats/sex/group). Test item was given through diet, in graduated doses of 150, 300, and 600 ppm, to three groups of male and female rats. The control group received basal diet. Male rats were treated for 43 days. Female rats were treated for two weeks prior to mating, variable time to conception, the duration of pregnancy and 14 days after delivery. The active ingredient concentration and homogeneity of the test item in diet were analysed once before the initiation of treatment and twice during the treatment period. Oestrous cycle length and pattern of female rats were evaluated by vaginal smears during pre-treatment of two weeks and daily from the beginning of the treatment period until evidence of mating. Rats were observed daily, twice, for mortality and morbidity, and once for visible clinical signs throughout the study period. Body weight of rats was recorded on the first day of treatment and the day of sacrifice. Body weight of male rats were recorded weekly throughout the treatment period. Body weights of female rats were recorded weekly during pre-mating and mating periods. Pregnant females were weighed on gestation days (GDs) 0, 7, 14, 20, within 24 hours of parturition, and post-coitum on day 25. Body weights of dams and pups were recorded on lactation days 0, 4, 7, and 14. Food consumption and test item intake was calculated during the treatment period. Pups were observed for sex, stillbirths, live birth, runts, gross anomalies, and measured for ano-genital distance (AGD) on post-natal day (PND) 0. Each litter was standardised on PND 4 to obtain nearly 4 male and 4 female pups. Rats were terminally sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination. Absolute organ weights were recorded and relative organ weights were calculated.

The results of diet formulation analyses were within the acceptable range of ±20% of the nominal concentration and the %CV was less than 20 which suggests that the prepared diet had an acceptable a.i. concentration and it was homogeneously prepared. Parents: No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 600 ppm dose level. The mean body weight gain, food consumption, and food efficiency of male rats, belonging to the 600 ppm dose group, was statistically significant decreased during treatment, when compared with that of the control group. The mean terminal body weight of female rats, belonging to the 600 ppm dose group, was statistically significant decreased, when compared with that of the control group. The mean body weight gain, food consumption, and food efficiency of female rats, belonging to the 600 ppm dose group, was statistically significant decreased during the pre-mating and lactation period, when compared with that of the control group. These decreases in mean body weight gain, food consumption, and food efficiency of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. Occational decreases in mean food consumption and food efficiency without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance. External examination of terminally sacrificed male and female rats, belonging to the 0, 150, 300, and 600 ppm dose groups did not reveal any abnormality. Greyish discolouration of liver of male and female rats, belonging to the 300 and 600 ppm dose groups, was observed during necropsy. Treatment related increases in mean absolute and relative liver weights of male rats, belonging to the 300 and 600 ppm dose groups was observed. Pups: Pup mortality was observed in litters, belonging to the 300 and 600 ppm dose groups. However, the body weight and body weight gain of pups, belonging to the 600 ppm dose group was comparable. Therefore, this could be considered as an incidental change. The mortality of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised and was not considered as treatment related. Pups mortality was also reflected in live birth and survival indices. The mean body weight and body weight gain of pups, belonging to the 600 ppm dose group, was statistically significant decreased during PNDs 7-14, when compared with that of the control group. This could be due to a decrease in mean food consumption and food efficiency of female rats of the 600 ppm dose group which might not produce sufficient milk for the pups. No treatment related differences were observed in litter size, sex ratio, AGD, and nipple retention of the pups. External and internal examination of the pups did not reveal any abnormality. A statistically significant increase in relative weights of thyroid with parathyroid was observed in male and female pups, belonging to the 600 ppm dose group.

Based on the results of the present study, the following dose levels are suggested for the extended one-generation reproductive toxicity study (cohort-1) of the test item in Wistar rats: 75, 150, and 300 ppm. The following NOAELs were determined based on the study:

- NOAEL systemic toxicity: 150 ppm, based on the gross pathology and greyish discoloration of the liver, both sexes (Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively)

- NOAEL reproductive toxicity: >600ppm, based on no adverse toxic effect observed, both sexes (Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively)

- NOAEL developmental toxicity: 300 ppm, based on the effect on the thyroid and parathyroid gland weight and the body weight ratios,  both sexes (Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively)

Effects on developmental toxicity

Description of key information

Jones 1996 - rat study - subchronic

- The NOAEL for parental toxicity is set 50 mg/kg body weight, highest dose tested.

- The NOAEL for developmental toxicity is 50mg/kg body weight, highest dose tested.

Christian 1999 - rat study - subacute

- The NOAEL for parental toxicity is set 50 mg/kg body weight, highest dose tested.

- The NOAEL for developmental toxicity is 50 mg/kg body weight/day, highest dose tested.

Latha 2018 - rabbit study - subacute

- The NOAEL for parental toxicity is set on 15 mg/kg body weight.

- The NOAEL for developmental toxicity is 15 mg/kg body weight/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04 Sep 2018 to 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0000145096
- Expiration date of the lot/batch: 03-01-2020
- Purity test date: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC)
- Solubility and stability of the test substance in the solvent/vehicle: Based on results of validation study, the test material formulations prepared at 1.0 and 250 mg/mL in the vehicle (Corn oil) are stable and resuspendable in the vehicle for 3 days when stored at room temperature at both the concentrations.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd., No.93, Solur, Thally Road, Anekal, Bengaluru-562106, India
- Age at study initiation: 7 months young adult, nulliparous and non-pregnant females at the initiation of the study and adult males (for mating purpose only).
- Weight at study initiation: G1: 2.975 ± 0.20, G2: 2.938 ± 0.21, G3: 2.984 ± 0.20, G4: 2.995 ± 0.20
- Housing: The rabbits were housed individually, except during cohabitation, when the females rabbits were cohabited with the males in 1:1 ratio in rabbit cages (approximate size: Length 65 cm x BWidth 65 cm x Height 45 cm) with shallow cage body and facilities for providing pelleted food (Stainless steel feed hopper) and drinking water in bottle fitted with sipper tube. The waste collection tray was changed daily (except on Sundays and public holidays). - Diet: Rabbit feed manufactured by Special Diets Services, Essex, England (Batch No.3439) was provided ad libitum in stainless steel feed hoppers to rabbits.
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier was provided ad libitum in polycarbonate bottles with stainless steel sipper tubes to rabbits.
- Acclimation period: After clinical examination for good health and suitability for the study, the rabbits were acclimatized for five days before initiation of mating. During the acclimatization period, all rabbits were observed at least once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 64-65, The relative humidity in the experimental rooms was calculated daily from dry and wet bulb temperature recordings.
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material dose formulations were prepared prior to treatment and used within the stability period. Required quantities of the test item were weighed separately for each dose group and transferred into separate mortars. The test item was triturated gently using a pestle to obtain a fine powder. A small portion of the vehicle (Corn oil) was added and the contents was triturated well to obtain a suspension. This suspension was transferred into a pre-marked beaker (Previously calibrated to the desired volume). The mortar and pestle was rinsed with the vehicle and the contents were transferred to the same pre-marked beaker. The suspension was made up to the intended volume with the vehicle. The homogeneity of the the test material dosing suspension during oral gavage was maintained by constant stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as vehicle for preparing the test material formulations in this study, as the same vehicle was used in pilot rabbit study without any effects.
- Concentration in vehicle: 0 mg/mL, 2.5 mg/mL, 7.5 mg/mL, 25 mg/mL
- Amount of vehicle: 2 mL/kg bw
-The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation. The actual volume administered was calculated based on the most recent body weight on first day of treatment (Gestation Day 6) and was adjusted according to the most recent body weights recorded till Gestation Day 28. Dose formulations were continuously stirred during dose administrations. The animals in the vehicle control group were handled and administered vehicle in an identical manner to the treatment groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and concentration analysis of test material, the prepared formulations were sampled at the initiation of treatment (GD 6) and at termination of treatment period (GD 28). Samples were drawn at each dose level (two samples from the top, middle and bottom layers) in duplicate sets. Similarly, samples only from middle layer was collected from vehicle control. The analyses was done using a validated analytical method. One set of samples was analyzed for concentration and other set of samples was kept as a backup at room temperature for reanalysis. These samples were discarded as the results of first set of samples were within the acceptable limits. Formulations were considered acceptable as the mean result for all layers was within ± 15% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: During the mating period, females were cohabited randomly with males in a 1:1 ratio
- Proof of pregnancy: After confirmation of mating by visual examination, the day was referred to as day 0 of pregnancy



Duration of treatment / exposure:
The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation.
Frequency of treatment:
Once per day
Duration of test:
All animals surviving until scheduled euthanasia were euthanised on day 29 post-coitum.
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
Total of 92 female rabbits were mated and allocated to a control group and 3 treatment groups (23 female rabbits/ group). Male rabbits were used only for mating.
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of Dose Levels and Dose Justification: A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with test material dosed at 5, 15 and 50 mg/kg bw/day along with the concurrent vehicle control group. The rabbits were treated once daily with test item formulations by oral gavage at a dose volume of 2 mL/kg body weight from GD 6 to 28 and observed for clinical signs and mortality. The test material at 5 mg/kg bw/day did not affect body weight, food consumption, and maternal and litter data parameters. At 15 mg/kg bw/day, body weight and food consumption were not affected, but there was reduction in mean fetal weights observed. Treatment at 50 mg/kg bw/day resulted in reduction in food consumption and there was reduction in uterine weight observed further causing reduction in the mean litter weights. These findings were considered test material treatment related. Gross evaluation of the placenta revealed no findings. No abnormality was noticed on external observations of fetuses at any of the doses tested. Based on the results of the dose range finding study and in consultation 0, 5, 15 and 50 mg/kg/day dose levels were selected for the definitive study.
Maternal examinations:
CLINICAL SIGNS AND MORTALITY:
- Clinical Signs and Mortality: All rabbits were observed for morbidity and mortality twice daily, i.e. once in the morning and once in the afternoon. Rabbits were observed for clinical signs during the treatment period of the study: pre dose and post dose (within 1- 2 hours of administration). On 20 September 2018, post dose observation was delayed by approximately 4.00 hrs. Aborted rabbits were necropsied, and gross observations observed were recorded.

BODY WEIGHT:
- All the females included in the study (G1 to G4) were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29. The intermittent body weight gain was calculated for GD 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 - 24, 24 - 27 and 27 - 29. Further the body weight gain for pre-treatment period (GD 0 to 6), treatment period (GD 6 to 28), and for entire gestation period (GD 0 to 29) was derived and statistically analyzed only for rabbits pregnant at caesarean section. The corrected body weight (carcass weight) was obtained by subtracting the unopened uterine weight from terminal body weight (body weight on GD 29). The corrected body weight gain was calculated by subtracting the body weight on GD 6 from corrected body weight.

FOOD CONSUMPTION:
- About 600 g of food (food input) was provided on GD 0. The left over food was recorded and replenished to about 600 g on GD 3, 6, 9, 12, 15, 18, 21, 24 and 27. Prior to terminal sacrifice, left over food was recorded on GD 29 of presumed gestation. There was no spillage of food noticed during the entire study period. Intermittent food consumption i.e GD 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 -24, 24 - 27 and 27 - 29 was calculated. Further, food intake for pre-treatment (GD 0 to 6), treatment (GD 6 to 29), and for entire gestation period (GD 0 to 29) was derived and statistically analyzed only for rabbits pregnant at caesarean section.

NECROPSY:
- Prior to caesarean section, random numbers were generated for coding to avoid bias during caesarean section and subsequent fetal evaluations. The animal code was written on the ear and the permanent accession number was striked out. On GD 29, all the rabbits were sacrificed by intravenous injection of sodium thiopentone. Gross pathological changes of all external and visceral organs of dams, including uterine contents were recorded.
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
- Other: gross evaluation of placenta
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The data on maternal body weight and food consumption, interval body weight changes, gravid uterine weight, body weight change corrected to gravid uterine weight were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney/Wilcoxon test pair wise comparisons of the treated groups with the control group was performed, when the group differences were significant. The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.
Indices:
All the indices can be found in the field 'Any other information on materials and methods, incl. tables'.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed at any of the doses tested during the study 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):
There was no change in mean maternal (corrected) body weights and (corrected) body weight gains during the different periods of gestation (GD 0-6; GD 6-29; GD 0-29) of the rabbits treated at 5 and 15 mg/kg/day compared to the vehicle control group. At 50 mg/kg/day there was statistically significant decrease in body weight gain during GD 6-29 (116%) and GD 0 – 29 (79%). In addition, there was a trend towards a decrease in mean maternal (corrected) body weight and corrected body weight gain, which was not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 5 and 15 mg/kg/day. Though not significant, there was a trend for a decrease in food consumption during different periods of gestation with stastistical significance during GD 21-24 at 50 mg/kg/day. The reduction in body weight and food consumption at 50 mg/kg/day is considered a treatment-related effect.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was a decrease in uterine weight (25%).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological findings in any of the treated groups except for a single occurrence of pale liver at 15 and 50 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
There were incidental occurrences of abortion in each of the low mid and high dose groups. One animal per group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was an increase in post implantation loss.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Two pregnant dams at the high dose of 50 mg/kg/day had complete resorptions.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was an increase in dams with resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were found in any of the dose groups
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The number of rabbits sacrificed at term were 23, 22, 22 and 22, respectively, of which 2, 2, 2 and 3 rabbits were non pregnant. The number of pregnant rabbits in the vehicle control, low, mid and high dose groups were 21, 20, 20 and 19, respectively.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
pre and post implantation loss
total litter losses by resorption
necropsy findings
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day, there was a significant reduction in mean fetal weights (-8.3%) at both the sexes (males and females) as compared to the vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The total number of fetuses were 135, 122, 118 and 94 at 0, 5, 15 and 50 mg/kg/day, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Information can be found in Table 1 in the field 'Any other information on results, incl. tables'.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related major abnormality was observed during external observations of the fetuses at any of the doses. Anomaly of fore limb flexed at wrist (moderate) was observed in one fetus of a litter with 5 fetuses at 50 mg/kg bw/day and in one fetus of a litter with 8 fetuses in the vehicle control group. These minor changes were considered incidental and not adverse as these observations commonly occur in animals of this test model. The incidence is attributed to random occurrence and not treatment related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related skeletal malformations observed in fetuses of dams treated up to 50 mg/kg/day. Variants and anomalies observed across vehicle and treated groups were comparable and are consistent with concurrent and historical data. The variants and anomalies were consistent with concurrent and historical data.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related major visceral malformations observed in fetuses of dams treated up to 50 mg/kg/day. Anomalies such as gall bladder hypoplastic in vehicle and test material treated groups, and bilobed gall bladder in the vehicle control group were observed. These findings were not considered adverse as these observations commonly occur in animals of this test model and the incidence of occurrence were consistent with concurrent or historical controls. At 50 mg/kg bw/day, visceral evaluation showed incidences of unilateral absence of kidney and ureter in one dam and unilateral dilated ureter with hydroneprosis in another dam. These findings (2/94 fetuses and 2/17 litters) were considered developmental anomalies.
Other effects:
no effects observed
Description (incidence and severity):
Gross evaluation of the placenta revealed no findings. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental Toxicity
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

For homogeneity and concentration analysis of test material it was found that the relative standard deviation of assay of top, middle and bottom layer (total 6 samples) was less than or equal to 10%.

Table 1. Summary of Litter data

Sex: female

G1

0

mg/kg/day

G2

5

mg/kg/day

G3

15

mg/kg/day

G4

50

mg/kg/day

Pregnant

N

21

20

21

19

Total number of fetuses

Mean 

N

Sum

6.4

21

135.0

6.0

20

119.0

5.9

20

118.0

5.5

17

94.0

% Male Fetuses

 

60.0

45.4

48.3

43.6

Dead

Sum

0

0

0

0

Live Male

Sum

81

55

57

41

Mean fetal [c] weight (M) (g)

Mean

SD

N

39.61

5.20

21

39.82

4.77

20

38.02

5.59

20

35.94

7.38

16

Live Female

Sum

54

67

61

53

Mean fetal [c1] weight (F) (g)

Mean

SD

N

40.22

6.24

21

39.83

4.78

20

37.13

4.22

20

36.03

7.26

17

Live fetuses

Sum

135

122

118

94

Mean fetal [c2] weight (both) (g)

Mean

SD

N

39.81

5.32

21

39.79

4.47

20

37.50

4.38

20

36.51*

7.21

17

[c] – Anova/Anova&Dunnett

{Covariate(s): Number of Live Male Fetuses}

[c1] - Anova/Anova&Dunnett

{Covariate(s): Number of Live Female Fetuses}

[c2] - Anova/Anova&Dunnett

{Covariate(s): Number of both Fetuses}; *=p<0.05

Table 2.            Summarized Details of Necropsy 

Parameters 

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

5

15

50

Total No. of rabbits / group

23

23

23

23

Duration of treatment 

GD 6 to 28 (total 23 days)

Caesarean section

(day of presumed gestation)

29

Number of rabbits sacrificed at caesarean section

23

22

22

22

Number of rabbits aborted

0

1

1

1

Number of rabbits non-pregnant at caesarean section

2

2

2

3

Number of rabbits pregnant at caesarean section

Number of rabbits with complete resorption

21

0

20

0

20

0

19

2

Number of litters examined

21

20

20

17

Total number of fetuses

135

122

118

94

 

 

 

 

 

Number of fetuses evaluated

 

 

 

 

External, visceral and skeletal

135

122

118

94

Number of fetus for decapitation

61

54

55

44

Number of intact fetuses

74

68

63

50

 

  Table 3. Summary of Maternal Body Weight of Pregnant Rabbits

Sex: Female

 

Day(s)

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Body Weight (kg)

0 [a]

Mean

SD

N

2.9731

0.2061

21

3.0035

0.2150

20

2.9701

0.2116

20

2.9880

0.1671

19

3 [a]

Mean

SD

N

3.0049

0.2171

21

3.0608

0.2312

20

2.9842

0.2261

20

3.0354

0.2011

19

6 [a]

Mean

SD

N

3.0279

0.2159

21

3.0947

0.2496

20

3.0148

0.2489

20

3.0541

0.1908

19

9[a]

Mean

SD

N

2.9963

0.2104

21

3.0166

0.2102

20

2.9551

0.2423

20

2.9587

0.1932

19

12 [a]

Mean

SD

N

3.0370

0.2183

21

3.0246

0.2512

20

2.9803

0.2381

20

2.9600

0.2331

19

15 [a]

Mean

SD

N

3.0925

0.2588

21

3.0756

0.2707

20

2.9968

0.2844

20

2.9672

0.1995

19

18 [a]

Mean

SD

N

3.1110

0.2546

21

3.1034

0.2673

20

2.9996

0.2799

20

2.9928

0.2294

19

21 [a]

Mean

SD

N

3.1415

0.2842

21

3.1024

0.2706

20

3.0086

0.2724

20

2.9965

0.2373

19

24 [a]

Mean

SD

N

3.1610

0.2899

21

3.1264

0.2659

20

3.0384

0.2709

20

3.0274

0.2524

19

27 [a]

Mean

SD

N

3.1636

0.3142

21

3.1256

0.2557

20

3.0443

0.2493

20

3.0205

0.2254

19

29 [a]

Mean

SD

N

3.1781

0.3041

21

3.1374

0.2504

20

3.0505

0.2179

20

3.0307

0.2454

19

 [a] – Anova & Dunnett(Log)

  

Table 4. Summary of Maternal Body Weight Gain of Pregnant Rabbits

Sex: Female

 

Day(s)

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Absolute Weight Gain (kg)

0 -> 3 [a]

Mean

SD

N

0.0318

0.0641

21

0.0573

0.0374

20

0.0140

0.0830

20

0.0475

0.0734

19

 

3 -> 6 [a1]

Mean

SD

N

0.0230

0.0527

21

0.0340

0.0395

20

0.0307

0.0494

20

0.0186

0.0431

19

 

6 -> 9 [a1]

Mean

SD

N

-0.0316

0.0778

21

-0.0781

0.0876

20

-0.0598

0.0743

20

-0.0953

0.0759

19

 

9 -> 12 [a1]

Mean

SD

N

0.0407

0.0855

21

0.0080

0.0967

20

0.0252

0.0856

20

0.0012

0.0840

19

 

12 -> 15 [a]

Mean

SD

N

0.0554

0.0740

21

0.0510

0.0720

20

0.0165

0.0925

20

0.0072

0.1001

19

 

15 -> 18 [a1]

Mean

SD

N

0.0185

0.0664

21

0.0279

0.0513

20

0.0028

0.0775

20

0.0256

0.0610

19

 

18 -> 21 [a]

Mean

SD

N

0.0306

0.0493

21

-0.0010

0.0447

20

0.0090

0.0757

20

0.0036

0.0461

19

 

21 -> 24 [a1]

Mean

SD

N

0.0194

0.0457

21

0.0240

0.0361

20

0.0298

0.0584

20

0.0309

0.0598

19

 

24 -> 27 [a1]

Mean

SD

N

0.0026

0.0701

21

-0.0007

0.0483

20

0.0059

0.0570

20

-0.0069

0.0486

19

 

27 -> 29 [a1]

Mean

SD

N

0.0145

0.0401

21

0.0118

0.0612

20

0.0062

0.0560

20

0.0102

0.0465

19

 

0 -> 6 [a]

Mean

SD

N

0.0548

0.0832

21

0.0912

0.0583

20

0.0447

0.0950

20

0.0661

0.0705

19

 

6 -> 29 [a1]

Mean

SD

N

0.1502

0.2006

21

0.0427

0.1361

20

0.0357

0.1844

20

-0.0234*

0.1614

19

 

0 -> 29 [a]

Mean

SD

N

0.2050

0.2326

21

0.1339

0.1331

20

0.0804

0.1645

20

0.0427*

0.1894

19

 

Corrected/adjusted Body weight (kg)

GD29 - gravid uterine. Wt [a2]

Mean

SD

N

2.8132

0.2704

21

2.7880

0.2443

20

2.7093

0.1993

20

2.7586

0.2638

19

 

Corrected/adjusted Body weight Gain

Corrected Bwt-GD6 [a1]

Mean

SD

N

-0.2147

0.1931

21

-0.3067

0.1271

20

-0.3055

0.1611

20

-0.2954

0.1820

19

 

 

[a] – Anova & Dunnett: *= p<0.05

[a1] – Anova & Dunnett(Rank): *= p<0.05

[a2] – Anova & Dunnett(Log)

  

Table 5. Summary of Maternal Data

Sex: Female

 

Day(s) Relative to mating

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Group size

GD 29

N

23

23

23

23

Pregnant

GD 29

N

21

20

20

19

Gravid Uterus (g)

GD 29 [a]

Mean

SD

N

364.9

79.9

21

349.4

62.5

20

341.3

64.4

20

286.1**

88.2

17

Number of CorporaLutea

GD 29 [k]

Mean

SD

N

8.2

1.4

21

8.1

1.4

20

8.4

2.0

20

8.2

1.9

17

Number of Implantation

GD 29 [k]

Mean

SD

N

7.0

1.7

21

6.6

1.2

20

6.6

1.8

20

6.4

2.0

17

With Early resorption

GD 29

Total

N

21

4

20

5

20

6

17

6

Number of Early Deaths

GD 29 [k]

Mean

SD

N

Sum

0.2

0.5

21

5.0

0.3

0.6

20

6.0

0.4

0.6

20

7.0

0.4

0.5

17

6.0

With Late Resorptions

GD 29

Total

N

21

4

20

4

20

6

17

7

Number of Late deaths

GD 29 [k]

Mean

SD

N

Sum

0.3

0.7

21

6.0

0.2

0.4

20

4.0

0.3

0.5

20

6.0

0.5

0.6

17

8.0

With Resorptions

GD 29

N

N-ve

N+ve

21

13

8

20

11

9

20

9

11

17

5

12

Total number of resorptions

GD 29 [f]

Mean

SD

N

Sum

Trend

0.5

0.8

21

11.0

   .

0.4

0.6

20

10.0

   .

0.7

0.7

20

13.0

0.1969.

0.8

0.6

17

14.0

0.0454*

Pre-imp Loss / Animal

GD 29 [k]

Mean

SD

N

1.24

0.94

21

1.50

1.05

20

1.85

1.25

20

1.82

1.63

17

[a] – Anova & Dunnett(Log)

[k] – Kruskal-Wallis & Wilcoxon 

[f] – Cochran Armitage, Chi-Squared & Chi squared - Pearson

 

 

Conclusions:
In this study, which conducted based on OECD TG 414 and under GLP conditions, prenatal developmental toxicity of the test material was evaluated in New Zealand White rabbits following daily administration by oral gavage at 0, 5, 15 and 50 mg/kg/day during gestation days 6 to 28. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity. Based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) are as follows:
- NOAEL maternal general toxicity is 15 mg/kg/day
- NOAEL maternal developmental toxicity is 15 mg/kg/day
- NOAEL developmental toxicity is 15 mg/kg/day
Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test material when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits.This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. Mated female rabbits were assigned to four groups with 23 animals in each group. The day on which mating had occurred was considered as gestation Day 0 (GD 0) for each individual female rabbit. Test material in vehicle (Corn oil) was administered at 0, 5, 15 and 50 mg/kg/day. The control group received the vehicle only. Dose formulation analysison initiation and termination of treatment periodindicated all samples were within 15% of the nominal concentrations. All rabbits were observed for clinical signs, morbidity and mortality, body weight changes and food consumption.Caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses.The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. The main findings of the study are presented below:

There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested except for incidental abortions in each of the, low, mid and high dose groups. Mean body weight gain at 50 mg/kg/day was lower compared to the vehicle control animals. Food consumption at 50 mg/kg/day was reduced during treatment period (GD 6 to 28). At 50 mg/kg/day there was a reduction in uterine weight, increase in post implantation loss and two dams with complete resorptions. At 50 mg/kg/day a significant reduction in litter weight was observed. Visceral evaluation at 50 mg/kg/day showed incidences (2/94 fetus) of unilateral absence of kidney and ureter and ureter dilated with hydronephrosis.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) for maternal and fetal developmental toxicity is 15 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 June - 30 November 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline on Detection of Toxicity to Reproduction for Medicinal Products, endorsed by the ICH Steering Committee
Principles of method if other than guideline:
The International Conference on Harmonisation (ICFI) Guideline on Detection of Toxicity to Reproduction for Medicinal Products, endorsed by the ICH Steering Committee Step 4 of the ICH process 24 June 1993. Section 4.1.2.was used, since there was no accepted protocol for such a study at that time. This guideline has also been issued as: JMOHW (Japanese Ministry of Health and Welfare): (Yakushin No. 470 of 7 July 1994).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CDBR VAF/Plus
Details on test animals and environmental conditions:
118 sexually mature female rats (Cr1 :CD®BR VAF/Plus strain), approximately 8 - 10 weeks, which were time-mated to males of the same strain, were received from Charles River UK Ltd, Manston Road, Margate, Kent. An additional 10 animals were supplied with batch A for health check purposes and subjected to routine macroscopic examination.
Initial group mean bodyweights within each batch (weight range 244 - 357 g).
Animal room temperature and relative humidity controls were set at 21 ± 2°C and 55 ± 10% respectively. Artificial lighting was controlled to give 12 hours continuous light and 12 hours continuous dark per 24 hours.
All animals had free access to tap water and pelleted SDS Laboratoiy Animal Diet No. 1.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test substance, AHTN, was administered as a solution in corn oil. The highest concentration formulation was prepared by mixing the test substance with vehicle. Lower concentrations were prepared by serial dilution. Control aniinals received the vehicle alone.
The formulations were prepared daily and used within 24 hours of preparation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Acetone extraction followed by High Performance Liquid Chromatography with UV detection.
The mean concentrations of test formulations analysed during the toxicity study were within 5% of the nominal concentration confirming the accuracy of preparation. The results also confirm that test formulations were true solutions and stable during ambient temperature storage for 24 hours, a period representing the maximum time from preparation to the completion of dosing.
Details on mating procedure:
The day of mating, as judged by the appearance of sperm in the vaginal smear or by the presence of a vaginal plug, was considered to be Day 0 of pregnancy.
F1: During the pre-mating period, F1 males and females animals were housed seperately and during the mating period, they were housed on the basis of one male to one female.
Duration of treatment / exposure:
The test substance, AHTN, was administered by gavage; only the F0 females received treatment. Treatment commenced on Day 14 of pregnancy and continued daily up to sacrifice on, or shortly after, Day 22 postpartum. The dosage volume was calculated for individual females on Days 14 and 16 of pregnancy according to bodyweight. Dose volumes were also adjusted according to bodyweight on Days 1, 7, 14 and 21 post partum.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
2, 6 and 20 mg/kg/day
Basis:

No. of animals per sex per dose:
112 animals with the highest weight gains were assigned to four groups i.e. 28 animals per dose group.
Control animals:
yes
Details on study design:
Duration of test: from day 14 of pregnancy up to sacrifice at weaning
Maternal examinations:
Occasional and isolated instances of salivation were noted after dosing amongst some treated animals but not controls.
There were no other clinical signs noted that were considered to be related to treatment.

There were no treatment-related effects on bodyweight gain or food intake following the start of treatment on Day 14 of pregnancy prior to parturition or on the pattern of bodyweight change during lactation.

The mean duration of pregnancy was not affected by treatment. There were no indications of any intergroup differences in the length of parturition.

There were no treatment-related changes in the incidence of minor abnormalities noted at post mortem examination of the females following weaning of their offspring.
Ovaries and uterine content:
Following the start of treatment on Day 14 of pregnancy, there were no treatment-related effects on the incidence of in utero losses.
Statistics:
Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on food consumption, bodyweight change, litter data, sex ratios, pre-weaning development, Post weaning behavioural tests, sexual maturation.

Dependent on the heterogeneity of variance between treatment groups, parametric tests were used to analyse the data, as appropriate. For litter data and pre-weaning development, the basic sampling unit was the litter and, due to the preponderance of non-normal distributions non parametric analyses were routinely used.

All significant (p ≤0.05) intergroup differences from the control are reported.
Where 75% or more of the values for a given variable were the same, a Fishers’ exact test was used, when considered necessary.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No treatment-related effects on mortality, body weight and food intake. The mean duration of pregnancy was also not affected. There were no treatment-related changes in the incidence of minor abnormalities noted at post mortem examination of the females following weaning of their offspring.
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects in birth weight. Mean values for litter size at birth were comparable for all groups. No evidence of any selective effect of treatment on survival of either sex.
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day
Basis for effect level:
other: No adverse toxic effect observed to the highest tested dose
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
no effects observed
Developmental effects observed:
no

There were no clinical signs noted amongst the F1 adults that were considered to be related to treatment of the F0 parent female.

Mean weekly bodyweight gain of F1 males and females from nominal Week 4 was unaffected by treatment of the F0 parent females.

There were no intergroup differences in the mean age of attainment or bodyweight on sexual maturation.

Treatment of parent females did not appear to impair the performance of their offspring on the Rotarod, Actimat or Passive Avoidance tests.

 

Treatment of the parent females had no adverse effect upon the mating performance of their offspring.

 

The incidence of macroscopic changes noted at autopsy of F1 adult males and females was low in all groups and considered to be unrelated to maternal treatment.

 

There were no macroscopic changes noted at post mortem examination of F2 pups that died or were sacrificed at weaning that were considered to be attributable to treatment of the F0 parent females.

Conclusions:
Treatment of the pregnant rat during the peri- and post natal period at dosages of AHTN up to (>)20 mg/kg bw/day was without adverse toxic effect. There were no effects on development of the foetus during the peri-natal phase of post natal growth, development, performance in specific behavioural tests and reproductive capacity of the offspring; exposure of the offspring was limited to possible in-utero transfer across the placenta during late pregnancy or via indirect transfer via the mothers milk.
Under the conditions of this study, the no-observable adverse effect level (NOAEL) on the pregnant and lactating rat and peri- and post natal development of the offspring was considered to be 20 mg/kg bw/day.
Executive summary:

The effect of AHTN on the development of the conceptus and offspring following exposure of the pregnant/lactating female during the peri-natal phase (from completion of organogenesis) to weaning. The primary intention of this study was to investigate whether exposure of the newborn to the test material via maternal milk resulted in any changes in Post natal development.

AHTN was administered at dosages of 0 (Control), 2, 6 or 20 mg/kg/day once daily by gavage to groups of 28 time-mated animals from Day 14 of pregnancy (end of organogenesis) through to weaning on Day 21 Post partum. The females were allowed to litter and rear their young to weaning (litters were standardised to 8 pups on Day 4 post partum). From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capability. The only exposure the F1 generation had to the test substance was in utero during the peri-natal phase or through any transfer in the milk of the lactating dams.

 

Apart from occasional, but not dosage-related, occurrences of salivation after dosing there were no apparent effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the foetus during the late pre-natal phase on post natal growth, development, performance in specific behavioural tests and reproductive capacity of the offspring.

Since no adverse effects were detected, no LOAEL can be established, and the NOAEL is higher than or equal to the highest dose tested, thus ≥20 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 - 30 january 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent to OECD guideline 414 (confirmed by Research Institute for Fragrance Materials). Study conducted by GLP accredited laboratory.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
AHTN in corn oil was daily administered through the gavage of rats during gestation. The dams were observed for toxicity and sacrificed for gross necropsy.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD BR VAF/Plus
Details on test animals and environmental conditions:
Groups of 25 Crl:CD®BR VAF/Plus® (Sprague-Dawley) female rats, supplied by Charles River Laboratories, Inc., Kingston, NY.
Female rats were individually housed except during the 5-day cohabitation period. All cage sizes and housing conditions were in compliance with the ‘Guide for the Care and Use of Labora tory Animals’ (Institute of Laboratory Animal Resources, 1996). Light cycles were automatically maintained at 12-h light:12-h dark. Rats were given ad libitum access to feed and water.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
AHTN was administered on days 7-17 of presumed gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis to check concentration and stability of AHTN in solution.
Details on mating procedure:
Dosage-range finding studies in rats were conducted at 100, 50, 25, 10 mg/kg per day. One animal from the 100 mg AHTN/kg per day dosage group was sacrificed in a moribund condition. Rats in the two highest AHTN dosage groups had reduced body weight gains.
Based on the results of these studies, dosages tested were 5, 15, 50 mg/kg per day.
Duration of treatment / exposure:
10 days (day 7-day 17 of gestation)
Frequency of treatment:
once daily
Duration of test:
20 days (of gestation)
Remarks:
Doses / Concentrations:
5, 15, 50 mg/kg/day
Basis:
other: The treatment period included days 7-17 of gestation (GD 7—17), and control and test articles were administered once daily by gavage at a dosage volume of 5 ml/kg, adjusted daily according to individual body weights recorded immediately before intubation.
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
Sex: female
Maternal examinations:
The dams were observed for signs of toxicity and body weights and feed intake were recorded. On day 20 of gestation, gross necropsy was performed.
Ovaries and uterine content:
The number of corpora lutea in the ovaries were recorded and the uteri were examined for pregnancy, number and distribution of implantations, live and dead foetuses and early and late resorptions. The placenta was also examined.
Fetal examinations:
All foetuses were weighed and examined for sex and gross external abnormalities. One half of the foetuses in each litter were examined for soft tissue alterations. The remaining foetuses were examined for skeletal alterations.
Statistics:
Statistical evaluations were performed using SAS programs. Clinical observations and other proportional data were analyzed by the Variance Test for Homogeneity of Binomial Distribution. Continuous data were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance or Dunnett’s Test. The Kruskal-Wallis Test along with Dunn’s Method of Multiple Comparisons or Fisher’s Exact Test was used if the Analysis of Variance was not appropriate.
Indices:
Gross necropsy parameters included: gross lesions and numbers of corpora lutea, implantations, live and dead foetuses and resorptions.
Foetus parameter: body weighed, sex and gross external alterations.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy rates were unaffected by the fragrances and ranged between 72 and 96% in the fragrance-treated groups, versus 84 and 100% in the two control groups. No abortions, premature deliveries or fragrance-related deaths occurred during the study.
Clinical signs, including urine-stained abdominal fur, excess salivation, red or brown substances on the forepaws, localized alopecia, tremors, chromodacryorrhea and dry faeces, were frequently increased in incidence in mid-dosage groups, and were significantly increased in the high-dosage groups, as compared with controls.
In the high-dosage group rats (50 mg/kg per day) localized alopecia and green or mottled livers were observed.
Dose-dependent, statistically significant reductions in maternal body weight gains and feed consumption occurred in all dosage groups with a significant rebound in body weight gains and feed consumption values in the high group.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The decreases in bodyweight were well within historical ranges and because of no clear dose relationship they were not considered relevant. No effects were observed on numbers of implantations, dead or live foetuses, resorptions or foetal sex ratios. There were no skeletal changes and all gross external, soft tissue or skeletal malformations in the foetuses were incidental and considered unrelated to the test article.
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: not selectively toxic to development; no adverse effects on development occurred
Abnormalities:
no effects observed
Developmental effects observed:
no

No abortions or premature deliveries occurred during the study. One non-pregnant 5 mg/kg/day dosage group rat was moribund sacrificed on presumed gestation day (DG) 11, because it had an injury of the snout/palate on or about DG 9, an event unrelated to the test article.

 

Significantly increased (p0.01) numbers of 50 mg/kg/day dosage group rats had localized alopecia, generally on the underside and back, but sometimes on the limbs. The 50 mg/kg/day dosage group also had a significant increase (p0.01) in the number of rats with colour changes in the liver (green or mottled green and dark red), findings attributable to the test article and not observed in any other dosage group.

 

The 5 mg/kg/day dosage group had a transient, small (p>0.05) reduction in maternal body weight gain, the 15 mg/kg/day dosage group had significantly reduced (p0.05) maternal body weight gain and the 50 mg/kg/day dosage group had significant maternal body weight loss (p0.01) on DGs 7 to 10. During the remainder of the dosage period, the 5 and 15 mg/kg/day dosage groups had comparable or slightly increased weight gains (DGs 12 to 15, p0.05, 15 mg/kg/day dosage group), and the 50 mg/kg/day dosage group had reduced weight gains (p0.01 on DGs 10 to 12 and 15 to 18), as compared with the control group values. As a result, the 50 mg/kg/day dosage group had significantly reduced (p0.01) body weight gain for the entire dosage period (calculated as DGs 7 to 18). After completion of the dosage period (DG5 18 to 20), groups administered 5 mg/kg/day and higher dosages of AHTN had dosage dependent increases in weight gains, as compared with the control group value (p0.05 to p0.01 in the 15 and 50 mg/kg/day dosage groups), rebound phenomena commonly observed in these types of studies after completion of the dosage period. These effects resulted in only the 50 mg/kg/day dosage group demonstrating significant reductions (p0.01) in weight gains for the entire period after the first dosage was administered (DGs 7 to 20) and for the entire pregnancy (DG5 0 to 20).

 

DG 0 maternal body weights were comparable following randomization and assignment to four dosage groups (Groups 1 through IV However, Groups II through IV gained significantly less weight (p0.01) during the pre-treatment period (DGs 0 to 7) than Group 1. These events were unrelated to the test article. On DG 7 (first treatment day), rats assigned to Groups II, III and IV had biologically comparable mean body weights (means ranged from 269.0 g to 265.2 g) that were reduced (Group II) or significantly reduced (Group III, p0.05; Group IV, p0.01), as compared with the control group (Group 1) value (275.2 g). The reduction in maternal body weight on DG 7 was considered unrelated to the test article. Maternal body weights were significantly reduced (p0.05) in Group II on DGs 8 through 10 and DG 12, as compared with the control group (Group 1) value. Maternal body weights were significantly reduced (p0.05 to p0.01) in Group III on DGs 7 through 14 and DG 16. Maternal body weights were significantly reduced (p0.0l) in Group IV on DGs 7 through 20.

 

Groups administered 5 mg/kg/day and higher dosages of AHTN had dosage dependent, significant (p0.05 to p≤0.01) reductions in absolute (g/day) feed consumption values for the entire dosage period (calculated as DGs 7 to 17). Relative (g/kg/day) feed consumption values tended to be reduced in the 5 mg/kg/day dosage group and were significantly reduced (p0.01) in the 15 and 50 mg/kg/day dosage groups on DG5 7 to 17. These effects of the test article reflected the cumulative effect of small reductions in absolute and relative feed consumption values in the 5 mg/kg/day dosage group and a reduced or significantly reduced (p≤0.05 to p≤0.01) absolute and relative feed consumption values throughout the dosage period in the 15 and 50 mg/kg/day dosage groups. As observed for body weight gain, the effects were most severe on DGs 7 to 10. Only the 50 mg/kg/day dosage group had a post-dosage (DGs 18 to 20) increase in relative feed consumption value, a rebound phenomenon. Absolute and relative feed consumption values were significantly reduced (p0.05 to p0.0l) in the 15 and 50 mg/kg/day dosage groups for the entire interval after the first dosage (DGs 7 to 17) and for the entire pregnancy (DGs 0 to 20).

 

The smaller, statistically significant (p0.05) reductions in foetal body weight parameters in the 5 and 15 mg/kg/day dosage groups were considered unrelated to the test article because they did not demonstrate dosage-dependency and were within historical ranges of the Testing Facility. The 50 mg/kg/day dosage group had statistically significant (p0.01) reductions in foetal body weight. These observations were not considered effects of the test article because, while the values were lower than in any other dosage group, the values were within ranges observed historically at the Testing Facility.

All other Caesarean-sectioning and litter parameters were unaffected by dosages of the test article as high as 50 mg/kg/day. No dosage-dependent, statistically significant or biologically important differences occurred in the litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early/late resorptions, percent resorbed conceptuses, or percent male foetuses. No dam had a litter consisting of only resorbed conceptuses, and there were no dead foetuses. All gross external, soft tissue or skeletal malformations or variations in the foetuses were considered unrelated to the test article.

Conclusions:
The maternal no-observable-adverse-effect-level (NOAEL) for AHTN is 5 mg/kg/day (minimal reductions in maternal body weight gain and feed consumption values occurred at this dosage). Maternal toxicity was evident in the 15 and 50 mg/kg/day dosage groups as adverse clinical and necropsy observations (50 mg/kg/day dosage group only) and biologically important, significant reductions in maternal body weight gains and absolute and relative feed consumption values.
The developmental NOAEL for AHTN is 50 mg/kg/day. Based on these data, the test article was not selectively toxic to development; no adverse effects on development occurred.
Executive summary:

AHTN was tested for developmental toxicity in rats (25/group, 3 groups/fragrance, 2 fragrances/corn oil control). Dosages tested was 5, 15, 50 mg/kg per day. These dosages exceeded multiples of the estimated maximal daily human dermal exposure. Treatment (gavage, 5 ml/kg) occurred on days 7-17 of gestation and Caesarean-sectioning on day 20 of gestation. Based on the results of these studies, none of the four fragrances tested were more toxic in the conceptuses than in the dams. Maternal NOAEL was 5 mg/kg per day (50 mg/kg per day caused clinical signs and reduced weight gain and feed consumption). Developmental NOAEL was 50mg/kg per day. No adverse effects on embryo-foetal viability, growth or morphology occurred at the highest dosages of AHTN (50 mg/kg per day). The results of this study indicate that under conditions of normal use, the test fragrances do not pose a risk to human conceptuses.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
OECD 414, Under GLP.
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

The objective of this study was to evaluate the prenatal developmental toxicity of the test material when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits.This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. Mated female rabbits were assigned to four groups with 23 animals in each group. The day on which mating had occurred was considered as gestation Day 0 (GD 0) for each individual female rabbit. Test material in vehicle (Corn oil) was administered at 0, 5, 15 and 50 mg/kg/day. The control group received the vehicle only. Dose formulation analysison initiation and termination of treatment periodindicated all samples were within 15% of the nominal concentrations. All rabbits were observed for clinical signs, morbidity and mortality, body weight changes and food consumption.Caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses.The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. The main findings of the study are presented below:

There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested except for incidental abortions in each of the, low, mid and high dose groups. Mean body weight gain at 50 mg/kg/day was lower compared to the vehicle control animals. Food consumption at 50 mg/kg/day was reduced during treatment period (GD 6 to 28). At 50 mg/kg/day there was a reduction in uterine weight, increase in post implantation loss and two dams with complete resorptions. At 50 mg/kg/day a significant reduction in litter weight was observed. Visceral evaluation at 50 mg/kg/day showed incidences (2/94 fetus) of unilateral absence of kidney and ureter and ureter dilated with hydronephrosis.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) for maternal and fetal developmental toxicity is 15 mg/kg/day.

  

The effect of AHTN on the development of the conceptus and offspring following exposure of the pregnant/lactating female during the peri-natal phase (from completion of organogenesis) to weaning. The primary intention of this study was to investigate whether exposure of the newborn to the test material via maternal milk resulted in any changes in Post natal development.

AHTN was administered at dosages of 0 (Control), 2, 6 or 20 mg/kg/day once daily by gavage to groups of 28 time-mated animals from Day 14 of pregnancy (end of organogenesis) through to weaning on Day 21 Post partum. The females were allowed to litter and rear their young to weaning (litters were standardised to 8 pups on Day 4 post partum). From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capability. The only exposure the F1 generation had to the test substance was in utero during the peri-natal phase or through any transfer in the milk of the lactating dams.

Apart from occasional, but not dosage-related, occurrences of salivation after dosing there were no apparent effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the foetus during the late pre-natal phase on post natal growth, development, performance in specific behavioural tests and reproductive capacity of the offspring.

Since no adverse effects were detected, no LOAEL can be established, and the NOAEL is higher than or equal to the highest dose tested, thus ≥20 mg/kg bw/day.

 

AHTN was tested for developmental toxicity in rats (25/group, 3 groups/fragrance, 2 fragrances/corn oil control). Dosages tested was 5, 15, 50 mg/kg per day. These dosages exceeded multiples of the estimated maximal daily human dermal exposure. Treatment (gavage, 5 ml/kg) occurred on days 7-17 of gestation and Caesarean-sectioning on day 20 of gestation. Based on the results of these studies, none of the four fragrances tested were more toxic in the conceptuses than in the dams. Maternal NOAEL was 5 mg/kg per day (50 mg/kg per day caused clinical signs and reduced weight gain and feed consumption). Developmental NOAEL was 50mg/kg per day. No adverse effects on embryo-foetal viability, growth or morphology occurred at the highest dosages of AHTN (50 mg/kg per day). The results of this study indicate that under conditions of normal use, the test fragrances do not pose a risk to human conceptuses.

Justification for classification or non-classification

Based on the available information classification for toxicity to reproduction is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.