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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in compliance with GLP and according to the OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
MATMD
IUPAC Name:
MATMD

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: males: 10-11 weeks; females: 10-11 weeks
- Weight at study initiation: males: 270 - 318 g; females: 184 - 219 g
- Housing: individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was ground to a fine powder and was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was mixed thoroughly using a homogenizer. The test item formulation was prepared once a week and stored at ambient temperature. Everyday 20 minutes before the dose administration the formulation was kept on a magnetic stirrer to ensure the homogeneity. Homogeneity of the test item in the vehicle was also maintained by keeping the prepared suspension on a magnetic stirrer during dose administration. The vehicle was also used as control item.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. The day of the vaginal sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 2 (first week of pre-mating period) and week 4 from all groups and stored between -15 and -35°C. Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and in the last week of the study (gestation/lactation) and stored between -15 and -35°C. On one of the weekly dose preparations the 8-day stability was performed. The samples were collected at 0 h and on day-8 from the high and low dose formulations and stored between -15 and -35°C. Each sample was retained twice. At the end of the treatment period all samples of dosing formulations were analysed in accordance with GLP.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.
The animals in the control group were handled in an identical manner to the test item treated group subjects and received PEG 400 using the same dose volume as used for the test item treated groups (5 mL/kg bw).
Frequency of treatment:
see above
Details on study schedule:
see above
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 30 or 60 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
The doses were selected on the basis of data from a Dose Range Finding Study and a 28-day study started 3 weeks prior to this study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1-2 times per day

DETAILED CLINICAL OBSERVATIONS: Yes
Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. The animal prematurely sacrificed was weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

WATER CONSUMPTION AND COMPOUND INTAKE: No data
Sperm parameters (parental animals):
For the testes a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS stained slides.
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattoo mark on paw. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on post-natal day 4.
Non-pregnant females were sacrificed on study day 26 from the day of evidence of mating.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Organs of the reproductive system (ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole)) and all organs showing macroscopic lesions of all adult animals were examined.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.
Postmortem examinations (offspring):
All surviving pups were sacrificed on PND 4. Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities (see also above).
Statistics:
Findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings. The evaluation included the relationship between the dosing of the test item and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.
The gestation length, pre coital interval, the number of live births and post implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, litter size and litter weights were summarized in tabular form.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights and food consumption were also presented as figures. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and are presented as percentage.
A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Gestation length, pre coital interval, number of live births and post implantation loss, number of implantations, corpora lutea, litter size and litter weights

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: The NOAEL was derived for both systemic toxicity of adult animals and the reproductive and developmental toxicity.

Results: F1 generation

General toxicity (F1)

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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
MATMD was tested in a study according to the OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) with groups of 10 male and 10 female Wistar rats with GLP compliance. The dose levels were 0, 15, 30 or 60 mg/kg bw.
The NOAEL for both systemic toxicity of adult animals (male and female) and the reproductive and developmental toxicity was 60 mg/kg bw/d.
Executive summary:

MATMD was tested in a study according to the OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) with groups of 10 male and 10 female Wistar rats with GLP compliance. The dose levels were 0, 15, 30 or 60 mg/kg bw.

Dose levels of > 15 mg/kg bw/d caused effects such as diarrhea, piloerection, salivation or nasal discharge in males and/or females, which also was noted in the control group. There was a tendency towards a reduced body weight gain and food intake in males at 60 mg/kg bw/d, which was assumed to be possible primary or secondary results of local irritative effects of the test item in the forestomach. There was no effect of toxicity noted at any dose levels for reproductive toxicity parameters. There were higher percent post implantation loss (without statistical significance) at >= 15 mg/kg bw/d, but this was within the historical control data range. Also taking into account the no. of corpora lutea, implantation sites and live pups (PND 0), which were comparable to the concurrent control group, the higher percent post implantation loss was not considered to be an adverse effect of treatment.

Thus, the NOAEL for both systemic toxicity of adult animals (male and female) and the reproductive and developmental toxicity was considered to be 60 mg/kg bw/d.