Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test guideline (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
8 January 2009 to 7 August 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to GLP and OECD guideline. Reliability 2 given due to the study being performed on a read-across substance. Read-across justification: The read-across substance (Gel All MD) is a close structural analogue of Gel All DX and comparison of the substances have shown them to be highly similar in phyisco-chemical properties and toxicological profile. It is therefore considered that the results from the read-across substance are valid for the assessment of Gel All DX. See justification of read-across document 'Justification of read-across for Gel All DX' (attachments section)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar Han™:HsdRccHan™:WIST strain rats, obtained from Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: (P) Males: Approximately 6 weeks old; Females: Approximately 10 weeks old.
- Weight at study initiation: (P) Males: 230 to 285 g; Females: 188 to 256 g.
- Housing: The animals were housed in groups of four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories UK Ltd., Oxon, UK), ad libitum.
- Water (e.g. ad libitum): Mains drinking water, ad libitum.
- Acclimation period: 7 days for males and 15 days for females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting, 12 hrs light/12 hrs dark.

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The formulations were shown to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The prepared formulations in the vehicle were within acceptable limits for the purpose of this study.
- Concentration in vehicle: 16.7 mg/ml, 50 mg/ml and 167 mg/ml.
- Amount of vehicle (if gavage): 6 ml/kg.
Details on mating procedure:
Males were paired with females on a one male: one female basis within each dose group, for a period of up to twenty-one days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary; The concentration of 4MDBS in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Materials and methods; The test material formulations were diluted with dimethylformamide to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Standard solutions of test material were prepared in dimethylformamide at a nominal concentration of 0.1 mg/ml.
The standards and samples were analysed by HPLC using the following conditions:
HPLC : Agilent Technologies 1050/1200, incorporating autosampler and workstation
Column : Sonoma 5μ C18 (250 x 4.6 mm id)
Mobile phase : Tetrahydrofuran:0.1% orthophosphoric acid (45:55 v/v)
Flow-rate : 1 ml/min
UV detector wavelength : 220 nm
Injection volume : 25 μl
Retention time : ~ 7.2 mins

The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
The test material formulations were sampled and analysed initially and then after storage at approximately 4ºC in the dark for 14 days. The test material formulations were sampled and analysed within 3 days of preparation.

Results; A range of standard solutions covering the concentration range 0 to 0.1541 mg/ml, were prepared and analysed. The detector response was shown to be linear up to 0.1541 mg/ml.
Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion; The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
Repeated oral administration for up to 19 consecutive weeks.

Administration by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats.

On completion of the dosing period all males were killed. Females were killed on Day 21 post partum.
Frequency of treatment:
Daily (except during littering/parturition for females).
Details on study schedule:
- Age at mating of the mated animals in the study: Male: Approximately 16 weeks old; Female: Approximately 12 weeks old.

Sequence of study:
i) Groups of 24 male and 24 female animals were treated at the appropriate dose level throughout the study (except during littering/parturition for
females).
ii) A vaginal smear was prepared daily for twenty-one days prior to pairing for all females.
iii) Following ten weeks of treatment for males and two weeks of treatment for females, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of twenty-one days.
iv) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
v) Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weights and clinical observations were also performed during this period.
vi) At Day 21 post partum, wherever possible, one male and one female offspring were selected for the evaluation of sexual maturation. At Day 21 post partum, all post partum females and remaining offspring were killed and examined macroscopically. Females which were not pregnant or failed to mate were also terminated.
vii) Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
Remarks:
Doses / Concentrations:
0 (control), 100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 animals per sex per dose (including control group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous repeat dose toxicity data.
Positive control:
No.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays.
- Cage side observations checked: Overt signs of toxicity, ill-health or behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations:
(Male) On Day 1 (prior to treatment) and then weekly until termination.
(Female)On Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0, 7, 14 and 21 post coitum and on Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly for each cage group for both sexes until pairing. Dietary intake for males was recorded weekly following the pairing phase until termination. Following confirmation of mating, dietary intake for females was recorded on Days 0 to 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4, 4 to 7, 7 to 14 and 14 to 21 of lactation.
- Food consumption for each animal determined and mean daily diet consumption: Yes, ratio of bodyweight change/dietary intake.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily, by visual inspection.

REPRODUCTION:
MATING
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. On a confirmation of positive evidence of mating, the males and mated females were housed individually.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female:
) Date of pairing/mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Oestrous cyclicity (parental animals):
Prior to pairing of females for the mating phase, a vaginal smear was taken daily for 21 days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The smears were examined microscopically and the stage of oestrous was recorded.
Sperm parameters (parental animals):
Parameters examined in male parental generations: Testes weight, epididymis weight, sperm count in testes, sperm concentration, motility, morphology and spermatid count in epididymides.


Litter observations:
PARAMETERS EXAMINED
On completion of parturition, the number of live and dead offspring was recorded. On Day 1 post partum, all surviving offspring within each litter were individually identified using ink tattoos on the feet (and tails).
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily.
iii) The sex of individual offspring was recorded on Days 1, 4, 7, 14, 21 post partum.
iv) Clinical condition of offspring from birth to weaning
v) Surface righting and ano-genital distance on Day 1.
vi) Individual offspring and total litter weights on Days 1, 4, 7, 14 and 21 post partum
vii) Necropsy findings of offspring

Selected F1 - Sexual Maturation:
Selected F1 offspring were observed for sexual development. For females the day of appearance of vaginal opening (separation of the labia) was recorded; for males the day of separation of the prepuce from the glans penis was recorded. The bodyweight for each individual at the time of sexual maturation was also recorded.


GROSS EXAMINATION OF DEAD PUPS:
Yes, for a full external and internal examination. Any macroscopic abnormalities were examined.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: On completion of the dosing period all males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- Maternal animals: Females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 21 post partum.

GROSS NECROPSY / ORGAN WEIGHTS
- Gross necropsy consisted of a full external and internal examination for all animals. Any macroscopic abnormalities were recorded.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium polysulphide solution where applicable (Salewski 1964).
(Organ weights) The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: left cauda epididymis, epididymides, kidneys, liver, ovaries, prostate, pituitary, seminal vesicles (with coagulating gland and fluids), testes and uterus (with cervix and oviducts).

HISTOPATHOLOGY
The following tissues were preserved from all males and females from each dose group, in buffered 10% formalin: coagulating gland, right epididymis (in Bouins fluid and then in 70% IMS after approximately forty-eight hours), ovaries, pituitary gland, prostate, seminal vesicles, right testis, uterus (with oviducts) and cervix, and vagina.
Microscopic examination was conducted by the Study Pathologist.

SEMEM ASSESSMENT:
At necropsy of adult males a semem assessment was performed.
Postmortem examinations (offspring):
SACRIFICE
All surviving offspring were terminated via carbon dioxide asphyxiation.

GROSS NECROPSY
- Gross necropsy consisted of a full external and internal examination. Any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights and offspring bodyweights were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dennett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.
Probability values (P) are presented as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (P) were calculated as follows:
P < 0.001 +++ --- ***
P < 0.01 ++ -- **
P < 0.05 + - *
P < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.
Reproductive indices:
Oestrous cycle
The stage of oestrus were classified according to the following criteria:
Dioestrus (D): Predominantly leucocytes present although some epithelial and cornified cells can be seen.
Proestrus (P): Predominantly epithelial cells, usually in significant numbers.
Early Oestrus (E1): Predominantly cornified cells, usually seen as small groups or isolated cells.
Late Oestrus (E2): Predominantly cornified cells usually seen as clumps of cells.
Metoestrus (M): Large numbers of leucocytes with discrete clumps of cornified cells.

The oestrous cycles are classified according to the following criteria:
Normal oestrous: The pattern of daily stages of oestrous show a four to five day cycle, which is generally repeated over 21 days.
Extended oestrous: The observation of a predominance of epithelial/cornified cells for more than two days for more than one oestrous cycle.
Extended dioestrous: The predominant cell type is the leucocyte for more than three consecutive days over more than one oestrous cycle.
Irregular cycle: An irregular length of oestrous cycle is observed over the 21 day evaluation period.
Acyclic - No evidence of an oestrous cycle is observed over the 21 day evaluation period.

Mating performance
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
Mating Index (%) = (Number of animals mated÷Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females÷Number of animals mated) x 100

Offspring viability indices:
Live Birth and Viability Indices
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1) x 100
Viability Index 2 (%) = (Number of offspring alive on Day 7 ÷ Number of offspring alive on Day 4) x 100
Viability Index 3 (%) = (Number of offspring alive on Day 14 ÷ Number of offspring alive on Day 7) x 100
Viability Index 4 (%) = (Number of offspring alive on Day 21 ÷ Number of offspring alive on Day 14) x 100
Viability Index 5 (%) = (Number of offspring alive on Day 21 ÷ Number of offspring alive on Day 1) x 100

Sex Ratio
Sex Ratio (% males) = (Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition).
There were no further unscheduled deaths during the study.

There were no clinical signs considered to be attributable to systemic toxicity in terminal kill animals. An episode of increased salivation was evident immediately before dosing for one male treated with 100 mg/kg/day on Day 51. Observations of this nature are commonly observed following the oral administration of a slightly unpalatable test material formulation, and in the absence of a similar effect seen at the high dose group the intergroup difference was not considered to represent a systemic effect of treatment. One female from all treatment groups showed instances of pallor of the extremities and one female treated with 100 mg/kg/day also showed hunched posture and pilo-erection. These observations were recorded around the time of parturition and were considered to be due to the littering process rather than a systemic effect of treatment.

The remaining clinical signs evident included generalised fur loss, staining of the external body surface, a damaged tail tip, chromodacryorrhea and scab formation. These were observed throughout the control and/or treatment groups and were considered to be low incidence findings occasionally observed in studies of this type and are unrelated to test material toxicity.

The male treated with 1000 mg/kg/day that was killed in extremis on Day 71 showed loss of righting and blink reflex, hypothermia, pallor of the extremities, hunched posture and lethargy.

BODY WEIGHT (PARENTAL ANIMALS)
There were no treatment-related effects detected in male bodyweight development. No adverse effects on bodyweight change were evident for treated females when compared to controls during the pre-mating, gestation and lactation phases of the study. Statisical analysis did not reveal any significant intergroup differences.

FOOD COMSUMPTION (PARENTAL ANIMALS)
No toxicologically significant effects were observed in males/females.
Males treated with 1000 mg/kg/day showed a statistically significant increase in food intake during Week 3, 6 and 16. An increase in dietary intake is not considered to be of toxicological importance.
Females treated with 1000 mg/kg/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in dietary intake is not considered to be of toxicological importance. No adverse effects on dietary intake were evident for treated females when compared to controls throughout lactation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No adverse effect on oestrous cycle was detected (in most of the cases, lasting 4 or 5 days).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No toxiciologically significant effects were detected.
Males treated with 1000 mg/kg/day showed a statistically significant reduction in progressive motility and testes spermatid count. In the absence of any histology correlates or an effect on fertility the intergroup differences were considered to be of no toxicological importance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No adverse effects on mating performance were detected for treated animals when compared to controls. Pre-coital intervals for the majority of paired animals were between 1 to 5 days. Two control pairs mated nine and thirteen days following pairing, two pairs from the 100 mg/kg/day dose group mated eight and eighteen days following pairing, two pairs treated with 300 mg/kg/day mated eight and nineteen days following pairing and four pairs treated with 1000 mg/kg/day mated seven, thirteen, sixteen and seventeen days following pairing. Positive evidence of mating was not confirmed for two pairs treated with 300 mg/kg/day, both of which however were pregnant and produced a litter.

There were no adverse effects detected on fertility or pregnancy status for treated animals when compared to controls. There was three non-pregnant female observed in the 100 mg/kg/day dose group, four non-pregnant females observed in the 300 mg/kg/day dose group and one non-pregnant female observed in the 1000 mg/kg/day dose group. Two females treated with 1000 mg/kg/day did show evidence of pregnancy however no offspring were observed to be born.

No treatment-related effects on the lengths of the gestation period were detected for treated females when compared to their concurrent controls. The majority of pregnant females showed gestation lengths between 21½ and 23 days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects were detected in the organ weights measured.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals.

One male treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day had small testes and epididymides at necropsy. The male treated with 300 mg/kg/day also had a turgid left testis. A further male treated with 300 mg/kg/day and one male treated with 100 mg/kg/day had small testes at necropsy. One female treated with 1000 mg/kg/day had a fluid filled sac encasing the right ovary. In the absence of any histology correlates detected in the testes, epididymides or ovaries the intergroup differences were considered to be of no toxicological significance. One female treated with 100 mg/kg/day had a fluid filled sac encasing the left kidney. A further female from this treatment group also had enlarged implantations on the left horn of the uterus. In the absence of a dose related response or any histology correlates the intergroup differences were considered to be of no toxicological importance. One control female, one female treated with 100 mg/kg/day, one female treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day had a nodule of the median lobe of the liver protruding through the diaphragm. This was considered to be due to physical damage
occurring during the necropsy procedure and not due to systemic toxicity.
A summary incidence of necropsy findings for males is given in Table 1 and for females is given in Table 2 as attached. Individual data are given in tables 3 and 4 as attached.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related microscopic abnormalities were detected.
A summary incidence of histopathological findings is given in Table 5 and Table 6 as attached. Individual data are given in tables 7 and 8 as attached.

OTHER FINDINGS (PARENTAL ANIMALS)
WATER COMSUMPTION
No toxicologically significant effects were observed in males/females.
Dose descriptor:
NOAEL
Remarks:
reproductive and developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant reproductive effects in any parameters examined at any dose levels tested.
Remarks on result:
other: Generation: P&F1 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
LITTER RESPONSES
With the exception of two pairs treated with 300 mg/kg/day, all animals mated. In total, twenty-three control females, nineteen 100 mg/kg/day females, nineteen 300 mg/kg/day females and nineteen females treated with 1000 mg/kg/day gave birth to a live litter and successfully reared young to Day 21 of age. Total litter losses were evident for one control female, one 100 mg/kg/day female, one 300 mg/kg/day and two females treated with 1000 mg/kg/day.

The following assessment of litter response is generally based on those litters reared to termination on Day 21 post partum, although data available for females showing a total litter loss has also been taken into consideration, where considered appropriate.


LITTER SIZE AND VIABILITY (OFFSPRING)
There were no treatment-related differences in the number of corpora lutea, implantation sites, pre-implantation losses or post implantation losses for treated animals when compared to controls. Litter sizes and the viability of the litters from treated animals were comparable to litters delivered by control dams. The ratio of males to females within each dose group was also similar for treated groups when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No clinically observable signs of toxicity were detected in offspring from the treated groups. The clinical observations observed throughout the control and treated groups were low incidence findings observed in studies of this type and were considered not to be related to treatment with the test material. The five total litter losses detected in a control female, a female treated with 100 mg/kg/day, a female treated with 300 mg/kg/day and a female treated with 1000 mg/kg/day were isolated and are occasionally observed in reproductive studies. Due to the presence of a total litter loss in the control group, these litter losses were considered to be unrelated to treatment.

BODY WEIGHT (OFFSPRING)
There were no adverse effects detected for litter weights or mean offspring bodyweights for offspring from treated animals when compared to offspring from control animals. Statistical analysis of the data did not reveal any significant intergroup differences.

SEXUAL MATURATION (OFFSPRING)
There were no treatment-related changes in the attainment of sexual maturation or ano-genital distance. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for both interim death and terminal kill offspring throughout the control and treated groups. The incidental findings observed throughout the control and treatment groups were occasionally observed low incidence findings in animals of the age employed and not considered to represent treatment-related changes in the affected tissues or organs.
Reproductive effects observed:
not specified
Conclusions:
The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.
Executive summary:

Introduction.

The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

Methods.

The test material was administered by gavage to three groups, each of twenty four male and twenty-four female Wistar Han™: HsdRccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Oestrous cycle assessment was performed daily for three weeks prior to mating.

After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. The ano-genital distance was recorded for all F1 generation offspring on Day 1 post partum.

All males were terminated following the completion of a successful mating. All surviving females and unselected offspring were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Selected offspring from each litter (where applicable) were evaluated for sexual maturation.

Results.

Mortality. One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition). There were no further unscheduled deaths during the study.

Clinical Observations. No clinically observable signs of toxicity were detected in terminal kill animals.

Bodyweight. No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout maturation, gestation or lactation.

Food Consumption. No adverse effect on food consumption was detected.

Water Consumption. No intergroup differences were detected.

Oestrous Cycle Assessment. There were no adverse effects on oestrous cycle assessments.

Mating. No adverse effects on mating performance was observed for treated animals when compared to controls.

Fertility & Pregnancy. There were no adverse effects of treatment on fertility or pregnancy rates between treated and control groups.

Gestation. No treatment-related effects were evident in the length of gestation for treated females when compared to controls.

Litter Responses. There was no adverse effect of treatment of the parent female on implantation rate, in-utero survival, litter size on Day 1 and subsequent offspring survival to weaning at dose levels of 100, 300 or 1000 mg/kg/day. Sex ratio of the offspring was essentially similar in all groups throughout lactation. There was no obvious adverse effect of treatment of the parent female on litter weights or offspring weight on Day 1 or subsequent bodyweight gain to weaning at 100, 300 or 1000 mg/kg/day.

Offspring Ano-genital Distances. No adverse effect was detected on ano-genital distance between treated and control groups.

Necropsy. No toxicologically significant effects were detected in terminal kill animals.

Organ Weights. No treatment-related effects were detected in the organ weights measured.

Sperm Analysis. No toxicologically significant effects were detected in the sperm parameters measured.

Histopathology. No treatment-related microscopic abnormalities were detected.

Conclusion. The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of

the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive

toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All relevant studies have been assigned a reliability 1 as GLP studies to OECD guidelines.
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 two studies used to assess reproductive toxicity are discussed below.

The read-across substance (Gel All MD) tested in the one generation reproduction study is a close structural analogue of Gel All DX and comparison of the substances have shown them to be highly similar in phyisco-chemical properties and toxicological profile. It is therefore considered that the results from the read-across substance are valid for the assessment of Gel All DX.

Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 421); Gel All DX:

The study is a valid investigation of the toxicological effects resulting from repeated oral-gavage admininstration of the test item Gel All DX to rats. Test item was administered in olive oil as vehicle at dose levels of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 46 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

Daily administration of the test item was well tolerated in males, females and pups, with no test item-related effects noted on any parameter measured (including mortality, clinical signs, food consumption, body weights, macroscopic/microscopic findings), at any dose level.

Reproduction and Breeding data:

Fertility, time course of mating, duration of gestation, corpora lutea count, number of implantation sites, post-implantation loss, litter size and post natal loss were unaffected by administration of the test item at any dose level.

Organ Weights:

No changes in absolute testes and epididymides weights or weights of these organs relative to body weight were noted at any dose level.

Oral Gavage One Generation Reproduction Study in the Rat (OECD 415); Gel All MD (read-across)

The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

The test material was administered by gavage to three groups, each of twenty-four male and twenty-four female Wistar Han™: HsdRccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study.Oestrous cycle assessment was performed daily for three weeks prior to mating.

After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. The ano-genital distance was recorded for all F1generation offspring on Day 1 post partum.

All males were terminated following the completion of a successful mating. All surviving females and unselected offspring were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Selected offspring from each litter (where applicable) were evaluated for sexual maturation.  

Results:

Mortality: One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition). There were no further unscheduled deaths during the study.

Clinical Observations: No clinically observable signs of toxicity were detected in terminal kill animals.

Bodyweight: No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout maturation, gestation or lactation.

Food consumption: No adverse effect on food consumption was detected.

Water Consumption: No intergroup differences were detected.

Oestrous Cycle Assessment.There were no adverse effects on oestrous cycle assessments.

Mating.No adverse effects on mating performance were observed for treated animals when compared to controls.

Fertility & Pregnancy.There were no adverse effects of treatment on fertility or pregnancy rates between treated and control groups.

Gestation.No treatment-related effects were evident in the length of gestation for treated females when compared to controls.

Necropsy.No toxicologically significant effects were detected in terminal kill animals.

Organ Weights.No treatment-related effects were detected in the organ weights (including epididymis and testes) measured.

Sperm Analysis.No toxicologically significant effects were detected in the sperm parameters measured.

Histopathology.No treatment-related microscopic abnormalities were detected in the reproductive organs examined.

The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.


Short description of key information:
Reproduction/developmental toxicity screening study (OECD 421): The NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.
One Generation Reproduction Study (on read-across substance Gel All MD): A NOAEL of 1000 mg/day was establised for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Justification for selection of Effect on fertility via oral route:
Toxicity to reproduction has been assessed based on two studies:
- a reproduction/developmental toxicity screening study (OECD 421)
- a one generation reproduction study (conducted on a read-across substance, Gel All MD)

Effects on developmental toxicity

Description of key information
Reproduction/developmental toxicity screening study (OECD 421): The NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.
One Generation Reproduction Study (on read-across substance Gel All MD): A NOAEL of 1000 mg/day was establised for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
8 January 2009 to 7 August 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to GLP and OECD guideline. Reliability 2 given due to the study being performed on a read-across substance. Read-across justification: The read-across substance (Gel All MD) is a close structural analogue of Gel All DX and comparison of the substances have shown them to be highly similar in phyisco-chemical properties and toxicological profile. It is therefore considered that the results from the read-across substance are valid for the assessment of Gel All DX. See justification of read-across document 'Justification of read-across for Gel All DX' (attachments section)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar Han™:HsdRccHan™:WIST strain rats, obtained from Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: (P) Males: Approximately 6 weeks old; Females: Approximately 10 weeks old.
- Weight at study initiation: (P) Males: 230 to 285 g; Females: 188 to 256 g.
- Housing: The animals were housed in groups of four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories UK Ltd., Oxon, UK), ad libitum.
- Water (e.g. ad libitum): Mains drinking water, ad libitum.
- Acclimation period: 7 days for males and 15 days for females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting, 12 hrs light/12 hrs dark.

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The formulations were shown to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The prepared formulations in the vehicle were within acceptable limits for the purpose of this study.
- Concentration in vehicle: 16.7 mg/ml, 50 mg/ml and 167 mg/ml.
- Amount of vehicle (if gavage): 6 ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary; The concentration of 4MDBS in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Materials and methods; The test material formulations were diluted with dimethylformamide to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Standard solutions of test material were prepared in dimethylformamide at a nominal concentration of 0.1 mg/ml.
The standards and samples were analysed by HPLC using the following conditions:
HPLC : Agilent Technologies 1050/1200, incorporating autosampler and workstation
Column : Sonoma 5μ C18 (250 x 4.6 mm id)
Mobile phase : Tetrahydrofuran:0.1% orthophosphoric acid (45:55 v/v)
Flow-rate : 1 ml/min
UV detector wavelength : 220 nm
Injection volume : 25 μl
Retention time : ~ 7.2 mins

The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
The test material formulations were sampled and analysed initially and then after storage at approximately 4ºC in the dark for 14 days. The test material formulations were sampled and analysed within 3 days of preparation.

Results; A range of standard solutions covering the concentration range 0 to 0.1541 mg/ml, were prepared and analysed. The detector response was shown to be linear up to 0.1541 mg/ml.
Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion; The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Details on mating procedure:
Males were paired with females on a one male: one female basis within each dose group, for a period of up to twenty-one days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
From: Day 0 Up To: Day 133 (males); Day 0 Up To: Day 21 post partum (females).
Frequency of treatment:
Daily (except during littering/parturition for females).
Duration of test:
Repeated oral administration for up to 19 consecutive weeks.

Administration by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats.

On completion of the dosing period all males were killed. Females were killed on Day 21 post partum.
Remarks:
Doses / Concentrations:
0 (control), 100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
nominal in vehicle
No. of animals per sex per dose:
24 animals per sex per dose (including control group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Age at mating of the mated animals in the study: Male: Approximately 16 weeks old; Female: Approximately 12 weeks old.

Sequence of study:
i) Groups of 24 male and 24 female animals were treated at the appropriate dose level throughout the study (except during littering/parturition for
females).
ii) A vaginal smear was prepared daily for twenty-one days prior to pairing for all females.
iii) Following ten weeks of treatment for males and two weeks of treatment for females, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of twenty-one days.
iv) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
v) Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weights and clinical observations were also performed during this period.
vi) At Day 21 post partum, wherever possible, one male and one female offspring were selected for the evaluation of sexual maturation. At Day 21 post partum, all post partum females and remaining offspring were killed and examined macroscopically. Females which were not pregnant or failed to mate were also terminated.
vii) Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
Maternal examinations:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays.
- Cage side observations checked: Overt signs of toxicity, ill-health or behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations:
(Male) On Day 1 (prior to treatment) and then weekly until termination.
(Female)On Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0, 7, 14 and 21 post coitum and on Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly for each cage group for both sexes until pairing. Dietary intake for males was recorded weekly following the pairing phase until termination. Following confirmation of mating, dietary intake for females was recorded on Days 0 to 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4, 4 to 7, 7 to 14 and 14 to 21 of lactation.
- Food consumption for each animal determined and mean daily diet consumption: Yes, ratio of bodyweight change/dietary intake.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily, by visual inspection.

REPRODUCTION:
MATING
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. On a confirmation of positive evidence of mating, the males and mated females were housed individually.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing/mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation



POST-MORTEM EXAMINATIONS: Yes
SACRIFICE
- Male animals: On completion of the dosing period all males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- Maternal animals: Females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 21 post partum.

GROSS NECROPSY / ORGAN WEIGHTS
- Gross necropsy consisted of a full external and internal examination for all animals. Any macroscopic abnormalities were recorded.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium polysulphide solution where applicable (Salewski 1964).
(Organ weights) The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: left cauda epididymis, epididymides, kidneys, liver, ovaries, prostate, pituitary, seminal vesicles (with coagulating gland and fluids), testes and uterus (with cervix and oviducts).

HISTOPATHOLOGY
The following tissues were preserved from all males and females from each dose group, in buffered 10% formalin: coagulating gland, right epididymis (in Bouins fluid and then in 70% IMS after approximately forty-eight hours), ovaries, pituitary gland, prostate, seminal vesicles, right testis, uterus (with oviducts) and cervix, and vagina.
Microscopic examination was conducted by the Study Pathologist.

SEMEM ASSESSMENT:
At necropsy of adult males a semem assessment was performed.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes
All surviving an dead offspring were terminated via carbon dioxide asphyxiation. A full external and internal examination. Any macroscopic abnormalities were recorded.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Others: The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, clinical condition, weight gain, sexual development and the bodyweight at the time of sexual maturation (for selected offspring).

Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights and offspring bodyweights were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dennett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.
Probability values (P) are presented as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (P) were calculated as follows:
P < 0.001 +++ --- ***
P < 0.01 ++ -- **
P < 0.05 + - *
P < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.
Indices:
Reproductive indices

Oestrous cycle
The stage of oestrus were classified according to the following criteria:
Dioestrus (D): Predominantly leucocytes present although some epithelial and cornified cells can be seen.
Proestrus (P): Predominantly epithelial cells, usually in significant numbers.
Early Oestrus (E1): Predominantly cornified cells, usually seen as small groups or isolated cells.
Late Oestrus (E2): Predominantly cornified cells usually seen as clumps of cells.
Metoestrus (M): Large numbers of leucocytes with discrete clumps of cornified cells.

The oestrous cycles are classified according to the following criteria:
Normal oestrous: The pattern of daily stages of oestrous show a four to five day cycle, which is generally repeated over 21 days.
Extended oestrous: The observation of a predominance of epithelial/cornified cells for more than two days for more than one oestrous cycle.
Extended dioestrous: The predominant cell type is the leucocyte for more than three consecutive days over more than one oestrous cycle.
Irregular cycle: An irregular length of oestrous cycle is observed over the 21 day evaluation period.
Acyclic - No evidence of an oestrous cycle is observed over the 21 day evaluation period.

Mating performance
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
Mating Index (%) = (Number of animals mated÷Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females÷Number of animals mated) x 100

(See 'Any other information on materials and methods incl. tables' section for the rest of Reproductive indices and Offspring viability indices.)
Historical control data:
Data were compared to the results observed.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
(Maternal systematic and reproductive toxicity)
CLINICAL SIGNS AND MORTALITY
One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition). There were no further unscheduled deaths during the study.

There were no clinical signs considered to be attributable to systemic toxicity in terminal kill animals. (An episode of increased salivation was evident immediately before dosing for one male treated with 100 mg/kg/day on Day 51. Observations of this nature are commonly observed following the oral administration of a slightly unpalatable test material formulation, and in the absence of a similar effect seen at the high dose group the intergroup difference was not considered to represent a systemic effect of treatment. One female from all treatment groups showed instances of pallor of the extremities and one female treated with 100 mg/kg/day also showed hunched posture and pilo-erection. These observations were recorded around the time of parturition and were considered to be due to the littering process rather than a systemic effect of treatment.

The remaining clinical signs evident included generalised fur loss, staining of the external body surface, a damaged tail tip, chromodacryorrhea and scab formation. These were observed throughout the control and/or treatment groups and were considered to be low incidence findings occasionally observed in studies of this type and are unrelated to test material toxicity.

The male treated with 1000 mg/kg/day that was killed in extremis on Day 71 showed loss of righting and blink reflex, hypothermia, pallor of the extremities, hunched posture and lethargy.

BODY WEIGHT
There were no treatment-related effects detected in male bodyweight development. No adverse effects on bodyweight change were evident for treated females when compared to controls during the pre-mating, gestation and lactation phases of the study. Statisical analysis did not reveal any significant intergroup differences.

FOOD COMSUMPTION
No toxicologically significant effects were observed in males/females.
Males treated with 1000 mg/kg/day showed a statistically significant increase in food intake during Week 3, 6 and 16. An increase in dietary intake is not considered to be of toxicological importance.
Females treated with 1000 mg/kg/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in dietary intake is not considered to be of toxicological importance. No adverse effects on dietary intake were evident for treated females when compared to controls throughout lactation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
No adverse effect on oestrous cycle was detected (in most of the cases, lasting 4 or 5 days).

REPRODUCTIVE FUNCTION: SPERM MEASURES
No toxiciologically significant effects were detected.
Males treated with 1000 mg/kg/day showed a statistically significant reduction in progressive motility and testes spermatid count. In the absence of any histology correlates or an effect on fertility the intergroup differences were considered to be of no toxicological importance.

REPRODUCTIVE PERFORMANCE
No adverse effects on mating performance were detected for treated animals when compared to controls. Pre-coital intervals for the majority of paired animals were between 1 to 5 days. Two control pairs mated nine and thirteen days following pairing, two pairs from the 100 mg/kg/day dose group mated eight and eighteen days following pairing, two pairs treated with 300 mg/kg/day mated eight and nineteen days following pairing and four pairs treated with 1000 mg/kg/day mated seven, thirteen, sixteen and seventeen days following pairing. Positive evidence of mating was not confirmed for two pairs treated with 300 mg/kg/day, both of which however were pregnant and produced a litter.

There were no adverse effects detected on fertility or pregnancy status for treated animals when compared to controls. There was three non-pregnant female observed in the 100 mg/kg/day dose group, four non-pregnant females observed in the 300 mg/kg/day dose group and one non-pregnant female observed in the 1000 mg/kg/day dose group. Two females treated with 1000 mg/kg/day did show evidence of pregnancy however no offspring were observed to be born.
No treatment-related effects on the lengths of the gestation period were detected for treated females when compared to their concurrent controls. The majority of pregnant females showed gestation lengths between 21½ and 23 days.

ORGAN WEIGHTS
No treatment-related effects were detected in the organ weights measured.

GROSS PATHOLOGY
There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals.

One male treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day had small testes and epididymides at necropsy. The male treated with 300 mg/kg/day also had a turgid left testis. A further male treated with 300 mg/kg/day and one male treated with 100 mg/kg/day had small testes at necropsy. One female treated with 1000 mg/kg/day had a fluid filled sac encasing the right ovary. In the absence of any histology correlates detected in the testes, epididymides or ovaries the intergroup differences were considered to be of no toxicological significance. One female treated with 100 mg/kg/day had a fluid filled sac encasing the left kidney. A further female from this treatment group also had enlarged implantations on the left horn of the uterus. In the absence of a dose related response or any histology correlates the intergroup differences were considered to be of no toxicological importance. One control female, one female treated with 100 mg/kg/day, one female treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day had a nodule of the median lobe of the liver protruding through the diaphragm. This was considered to be due to physical damage
occurring during the necropsy procedure and not due to systemic toxicity.

A summary incidence of necropsy findings for males is given in Table 1 and for females is given in Table 2 as attached. Individual data are given in tables 3 and 4 as attached.

HISTOPATHOLOGY
No treatment-related microscopic abnormalities were detected.
A summary incidence of histopathological findings is given in Table 5 and Table 6 as attached. Individual data are given in tables 7 and 8 as attached.

WATER COMSUMPTION
No toxicologically significant effects were observed in males/females.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER RESPONSES
With the exception of two pairs treated with 300 mg/kg/day, all animals mated. In total, twenty-three control females, nineteen 100 mg/kg/day females, nineteen 300 mg/kg/day females and nineteen females treated with 1000 mg/kg/day gave birth to a live litter and successfully reared young to Day 21 of age. Total litter losses were evident for one control female, one 100 mg/kg/day female, one 300 mg/kg/day and two females treated with 1000 mg/kg/day.

The following assessment of litter response is generally based on those litters reared to termination on Day 21 post partum, although data available for females showing a total litter loss has also been taken into consideration, where considered appropriate.

LITTER SIZE AND VIABILITY
There were no treatment-related differences in the number of corpora lutea, implantation sites, pre-implantation losses or post implantation losses for treated animals when compared to controls. Litter sizes and the viability of the litters from treated animals were comparable to litters delivered by control dams. The ratio of males to females within each dose group was also similar for treated groups when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS
No clinically observable signs of toxicity were detected in offspring from the treated groups. The clinical observations observed throughout the control and treated groups were low incidence findings observed in studies of this type and were considered not to be related to treatment with the test material. The five total litter losses detected in a control female, a female treated with 100 mg/kg/day, a female treated with 300 mg/kg/day and a female treated with 1000 mg/kg/day were isolated and are occasionally observed in reproductive studies. Due to the presence of a total litter loss in the control group, these litter losses were considered to be unrelated to treatment.

BODY WEIGHT
There were no adverse effects detected for litter weights or mean offspring bodyweights for offspring from treated animals when compared to offspring from control animals. Statistical analysis of the data did not reveal any significant intergroup differences.

SEXUAL MATURATION
There were no treatment-related changes in the attainment of sexual maturation or ano-genital distance. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were detected for both interim death and terminal kill offspring throughout the control and treated groups. The incidental findings observed throughout the control and treatment groups were occasionally observed low incidence findings in animals of the age employed and not considered to represent treatment-related changes in the affected tissues or organs.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.
Executive summary:

Introduction: The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

Methods: The test material was administered by gavage to three groups, each of twenty four male and twenty-four female Wistar Han™: HsdRccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Oestrous cycle assessment was performed daily for three weeks prior to mating.

After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. The ano-genital distance was recorded for all F1 generation offspring on Day 1 post partum.

All males were terminated following the completion of a successful mating. All surviving females and unselected offspring were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Selected offspring from each litter (where applicable) were evaluated for sexual maturation.

Results.

Mortality. One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition). There were no further unscheduled deaths during the study.

Clinical Observations. No clinically observable signs of toxicity were detected in terminal kill animals.

Bodyweight. No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout maturation, gestation or lactation.

Food Consumption. No adverse effect on food consumption was detected.

Water Consumption. No intergroup differences were detected.

Oestrous Cycle Assessment. There were no adverse effects on oestrous cycle assessments.

Mating. No adverse effects on mating performance was observed for treated animals when compared to controls.

Fertility & Pregnancy. There were no adverse effects of treatment on fertility or pregnancy rates between treated and control groups.

Gestation. No treatment-related effects were evident in the length of gestation for treated females when compared to controls.

Litter Responses. There was no adverse effect of treatment of the parent female on implantation rate, in-utero survival, litter size on Day 1 and subsequent offspring survival to weaning at dose levels of 100, 300 or 1000 mg/kg/day. Sex ratio of the offspring was essentially similar in all groups throughout lactation. There was no obvious adverse effect of treatment of the parent female on litter weights or offspring weight on Day 1 or subsequent bodyweight gain to weaning at 100, 300 or 1000 mg/kg/day.

Offspring Ano-genital Distances. No adverse effect was detected on ano-genital distance between treated and control groups.

Necropsy. No toxicologically significant effects were detected in terminal kill animals.

Organ Weights. No treatment-related effects were detected in the organ weights measured.

Sperm Analysis. No toxicologically significant effects were detected in the sperm parameters measured.

Histopathology. No treatment-related microscopic abnormalities were detected.

Conclusion: The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All relevant studies have been assigned a reliability 1 as GLP studies to OECD guidelines.
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 two studies used to assess reproductive toxicity are discussed below.

The read-across substance (Gel All MD) tested in the one generation reproduction study is a close structural analogue of Gel All DX and comparison of the substances have shown them to be highly similar in phyisco-chemical properties and toxicological profile. It is therefore considered that the results from the read-across substance are valid for the assessment of Gel All DX.

Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 421); Gel All DX:

See fertility section for details on study design.

 

Daily administration of the test item was well tolerated in males, females and pups, with no test item-related effects noted on any parameter measured (including mortality, clinical signs, food consumption, body weights, macroscopic/microscopic findings), at any dose level.

Litter Data - F1 Pups

Findings at First Litter Check and During Lactation

No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

Pups sex ratio was not affected by exposure to the test item at any dose level.

Pup Weights to Day 4 Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

No test item-related findings were noted in pups at necropsy at any dose level.

Oral Gavage One Generation Reproduction Study in the Rat (OECD 415); Gel All MD (read-across):

See fertility section for details of study design.

Results

Litter Responses. There was no adverse effect of treatment of the parent female on implantation rate,in-uterosurvival, litter size on Day 1 and subsequent offspring survival to weaning at dose levels of 100, 300 or 1000 mg/kg/day.

Sex ratio of the offspring was essentially similar in all groups throughout lactation.

There was no obvious adverse effect of treatment of the parent female on litter weights or offspring weight on Day 1 or subsequent bodyweight gain to weaning at 100, 300 or 1000 mg/kg/day.

Sexual Maturation of the F1 Generation: There were no treatment-related changes in the attainment of sexual maturation.

Offspring Ano-genital Distances. No adverse effect was detected on ano-genital distance between treated and control groups.

Necropsy.No toxicologically significant effects were detected in offspring animals.

A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.


Justification for selection of Effect on developmental toxicity: via oral route:
See effects on fertility

Justification for classification or non-classification

Two valid studies are available to assess for classification for reproductive toxicity.

The OECD 421 Reproduction/Developmental Toxicity Screening Test is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats.

 

Daily administration of the test item was well tolerated in males, females and pups, with no test item-related effects noted on any parameter measured, at any dose level.

 

Reproductive performance was no affected by the treatment. Mating performance, fertility, corpora lutea count, implantation rate and post-implantation loss, duration of gestation, litter size and pups condition were similar in all groups.

Therefore, based on the results of the OECD 421 study there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development at the highest dose level. The substance is therefore not classified for reproductive toxicity based on this study.

In a one-generation reproduction study (on a read-across substance) administration of the test material did not affect mating performance, fertility or reproductive performance in any animals. There were no treatment-related effects detected in litter size or viability, growth or development of litters from treated animals and post-mortem finding of offspring did not reveal any treatment related changes at the dose levels assessed in the study.

There is no evidence that the tested substance has an adverse effect on reproduction or development at the highest dose level (1000 mg/kg bw/day). The substance is therefore not classified for reproductive toxicity based on this study.