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Toxicological information

Toxicity to reproduction

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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
Study period:
11-July-2006 - 30-Nov-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Dept. of Health, UK
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Mercaptoacetic acid, monoester with propane-1,2,3-triol
EC Number:
250-264-8
EC Name:
Mercaptoacetic acid, monoester with propane-1,2,3-triol
Cas Number:
30618-84-9
Molecular formula:
C5H10O4S
IUPAC Name:
mercaptoacetic acid, monoester with propane-1,2,3-triol
Constituent 2
Reference substance name:
GMT 80
IUPAC Name:
GMT 80
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): Glycerol Thioglycolate 80 %
- Analytical purity: mean 80.6% (w/w)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 280 - 379 g; Females: 192 - 260 g
- Housing: All animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male: one female basis.
Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 28 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 % relative humidity
- Air changes (per hr): 15 x / h
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- The test material was administered by gavage to three groups each of ten male and ten female rats, for up to fifty-four consecutive days, at dose levels of 15, 50 and 150 mg/kg/day.
- A control group of ten males and ten females was dosed with vehicle alone (Deoxygenated and demineralised water).
- Two recovery groups, each of five males and five females, were treated with the high dose (150 mg/kg/day) or the vehicle alone for forty-three consecutive days and then maintained without treatment for a further fourteen days.
Details on mating procedure:
- On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days and smearing commenced.
- Following evidence of mating using vaginal smearing, the males were returned to their original cages and females were transferred to individual cages.
- On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
- Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Groups of ten males and females were treated daily. On Day 15 they were paired for a maximum of 14 days, pregnant females were allowed to give birth and maintain their offsprings until Day 5 post partum.
Frequency of treatment:
Each day
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
15, 50 and 150 mg/kg bw/day by gavage
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
none

Examinations

Parental animals: Observations and examinations:
- Clinical Observations: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after 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 (except for females during parturition where applicable). During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.

- Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

- Behavioural Assessments: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

- Functional Performance Tests: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions.

- Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Startle reflex, Tail pinch, Blink reflex, Finger approach

- Bodyweight: Individual bodyweights were recorded on Day l (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 postpartum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

- Food Consumption: During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

- Water Consumption: Water intake was observed daily by visual inspection of water bottles for any overt change.

- Laboratory Investigations: Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group on Day 14 (day prior to pairing). Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

- Urinalytical investigations were performed on five males selected from non-recovery test and control group during the final week of treatment and on five males selected from each recovery group during the final week of the fourteen day treatment-free period.

- Haematology: The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Het), Erythrocyte indices, Total leucocyte count (WBC), Differential leucocyte count, Platelet count (PLT), Reticulocyte count

- Blood Chemistry: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Calcium (Ca++), Glucose Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili)

- Urinalysis: The following parameters were recorded on collected urine: Volume, Ketones, Specific Gravity, Bilirubin, pH, Urobilinogen, Protein, Reducing Substances, Glucose, Blood.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- Testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of all pups/litter

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Number of offspring born
- Number and sex of offspring alive recorded daily and reported on Day 1 and 4 postpartum
- Clinical condition of offspring from birth to Day 5 postpartum
- Individual offspring and litter weights on Day 1 and 4 postpartum
- All live offspring were assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum.


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

ORGAN WEIGHTS:
- These reproductive organs were weighed from all animals that were killed at the end of the study: Ovaries, testes, epididymides
- The following organs, removed from five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys, thymus

HISTOPATHOLOGY:
- From all animals: Ovaries, pituitary, prostate, coagulating gland, seminal vesicles, epididymides, testes, uterus/cervix
- From five selected males and females of each group that were killed at the end of the study: Adrenals, ovaries, aorta (thoracic), pancreas,bone & bone marrow (femur including stifle joint), pituitary, bone & bone marrow (stemum), prostate, brain (including cerebrum, cerebellum and pons), oesophagis, caecturi, rectum, coagulating gland, salivary glands (submaxillary), colon, sciatic nerve, duodenum, seminal vesicles, epididymides, skin (hind limb), eyes, spinal cord (cervical), gross lesions, spleen, heart, stomach, ileum, thyroid/parathyroid Jejunum, trachea, kidneys, testes, liver, thymus, lungs (with bronchi), urinary bladder, lymph nodes (cervical and mesenteric), uterus/cervix, mammary gland, vagina, muscle (skeletal)
Postmortem examinations (offspring):
Not examined
Statistics:
- Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight),
weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional
performance data 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 Dunnett’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
landmark developmental markers.
- The haematology variable basophils was not analysed since consistently greater than 30% of the
data were recorded as the same value.
Reproductive indices:
Pre-coital interval and fertility indices were calculated
Offspring viability indices:
Live birth and viability indices were calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY: There were five interim deaths during the study, these were confined to females treated with 150 mg/kg/day. One female (number 76) died during the maturation phase on Day 10. A second female (number 73) was killed in extremis on Day 23 (Day 7 of gestation). Two females were found dead on their Day 22 of gestation (Day 40 of the main study for female 80 and Day 41 of the main study for female 75), The final of the five females (number 72) was killed in extremis on it’s Day 1 of lactation phase (Day 41 of main study).

CLINICAL SIGNS: The only toxicological significant observations were detected for the two interim death females that were killed in extremis (numbers 73 and 72), these included incidents of tip-toe gait, hunched posture and pilo-erection. One of the females (number 73) also had red/brown staining around the mouth. The deterioration of the females’ condition subsequently led to their termination. No toxicologically significant findings were detected for animals of either sex after the treatment period, or for animals of either sex after the fourteen-day recovery period.

BODY WEIGHT AND WEIGHT GAIN: Females treated with 150 mg/kg/day showed a reduction in group mean bodyweight and bodyweight gain between Day 0 and Day 7 of gestation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was reduced for females treated with 150 mg/kg/day
during Days 0 to 7 of gestation when compared to controls although statistical significance was not achieved.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No overt intergroup differences were detected for all animals after the
treatment period


HAEMATOLOGY: No treatment-related effects were detected for the haematological parameters investigated


CLINICAL CHEMISTRY: No treatment-related intergroup differences in blood chemical parameters were detected


URINALYSIS: No toxicologically significant effects were detected in treated animals when compared to controls


NEUROBEHAVIOUR: No treatment-related effects were detected for animals of either sex from all treatment groups after the treatment period. Formal behavioural assessments were not carried out for recovery group animals.


ORGAN WEIGHTS: No treatment-related intergroup differences in organ weights were detected for all treated animals after the treatment period


HISTOPATHOLOGY: There were no treatment-related microscopic changes detected for the pituitary or reproductive tissues examined for terminal kill animals after the treatment period.here were five unscheduled deaths during the course of the study. Three females were found dead and two were killed in extremis, all five females were from the 150 mg/kg/day treatment group. With the possible exception of pyometria reported for femal number 72 and focal gastric ulceration for female 73, factors contributing to the death or moribundity in these animals were not evident from histopathological evaluation




Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: clinical signs; mortality; body weight

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- No intergroup differences were observed

CLINICAL SIGNS (OFFSPRING)
- There were no clinically observable signs of test material toxicity detected in offspring from treated animals.

BODY WEIGHT (OFFSPRING)
- Offspring weight at Day 1 of lactation was reduced in the 150 mg/kg/day dose group. This was due to a single dam (number 72) and litter that was killed in extremis on Day 1 of lactation due to the deterioration of the physical health of the dam. The poor condition of the dam led to the reduced bodyweight detected in the animal’s offspring, thus, leading to the reduction in group mean bodyweight for offspring on Day 1 lactation. The offspring bodyweight for the remaining 150 mg/kg/day litters were comparable to controls at Day 4 of lactation. The actual bodyweight and the offspring bodyweight change between Day 1 and 4 of lactation for the remaining litters in the 150 mg/kg/day treatment group were comparable to controls, therefore, this slight difference in offspring bodyweight was considered to be a result of the poor condition of a single dam in the 150 mg/kg/day treatment group and not a representation of reproductive toxicity.

SEXUAL MATURATION (OFFSPRING)
- No treatment-related effects on offspring development were detected.

OTHER FINDINGS (OFFSPRING)
- No intergroup differences were observed for litter size or sex ratio
- No treatment related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of GMT 80% to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/d, resulted in no treatment related effects for the males treated with 150 mg/kg bw/d but did result in the death of five females treated with 150 mg/kg bw/d. The NOAEL for systemic toxicity was considered to be 150 mg/kg bw/d for males and 50 mg/kg bw/d for females. The NOAEL for reproductive effects in the males is also 150 mg/kg bw/d as assessed by the absence of adverse effects on fertility or on testicular weight and histopathology. Due to the high matemal mortality and toxicity at 150 mg/kg bw/d in the females, which precludes reproductive assessment at that dose level, the NOAEL for reproductive toxicity in females is 50 mg/kg bw/d.