Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-12 to 2018-08-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-12 to 2018-08-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészégügyi Intézet
Limit test:
no
Species:
rat
Strain:
other: Han:WIST of Wistar origin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 85-90 days; Females: 85-90 days
- Weight at study initiation: Males: 315-396 g; Females: 190-230 g
- Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females: individually
Males after mating: 2 animals/cage
Recovery animals: 2 or 3 animals of the same sex/cage
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice provided ad libitum
- Water: tap water provided ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod: Artificial light, from 6 a.m. to 6 p.m
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Butylmonoglycol sulphate, Na-salt was formulated in the vehicle (distilled water) at concentrations of 20, 60 and 200 mg/mL in the formulation laboratory of the Test Facility daily.

VEHICLE
- Amount of vehicle: Control animals were handled in an identical manner to the test groups receiving vehicle (5 mL/kg bw).
- Lot/batch no. (if required): 1801-5512; 1801-5512; 1803-5501
Details on mating procedure:
- M/F ratio per cage: 1:1 mating
- Length of cohabitation: Females remained with the same male until copulation occurred.
- Proof of pregnancy: vaginal plug or sperm referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study.
Duration of treatment / exposure:
49 days (male animals) or 50-56 days (female animals); the day of first treatment is considered as Day 0 of examination.
Frequency of treatment:
7 days/week basis, every day at a similar time (±2 hours)
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
12 animals per sex per dose
5 animals per sex for control and high dose group as recovery group
Control animals:
yes, concurrent vehicle
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made on parental animals once a day, after the administration at approximately the same time. Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.
- Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy (treatment and recovery periods). Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10. Body weight was measured on day of necropsy for female animals subjected to organ weighing. Animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment period.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week (Day 49 (corresponding to PND 9-12 for dams) before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.

Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smears were also prepared on the day of the necropsy. Vaginal smears were stained with 1% aqueous methylene blue solution and were examined with a light microscope.
Sperm parameters (parental animals):
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were comparable in quantity and morphologically in the testes of investigated control and test item treated animals.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), and presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes; dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities. Thyroid gland was preserved from one male and one female pup per litter.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving male animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP after the optionally extended post-mating period on Day 49
- Maternal animals: All surviving female animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP. Dams were sacrificed either on post-partum day 14 or shortly thereafter (Days 50, 51, 52, 54, 55 or 56) or shortly after post-partum day 13 (Day 54).

GROSS NECROPSY
- All animals were subjected to a full detailed gross necropsy. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- All surviving F1 offspring were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”) and were subjected to gross necropsy on postnatal day 13 or shortly thereafter.

GROSS NECROPSY
- Gross necropsy consisted of an examination for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
At terminal sacrifice, the thyroids from 1 male and 1 female pup per litter were preserved. If possible. In addition, blood samples were collected for determination of serum levels of thyroid hormones (T4 and TSH) from at least two pups per litter on post-natal day 4 (if it was feasible). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk.
Statistics:
The statistical evaluation of appropriate data (marked † above) was performed with the statistical program package SPSS PC+4.0.

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.

Where no significant heterogeneity was detected, an one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi² test was performed if feasible.

For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
Reproductive indices:
Copulatory index: Measure of animals’ ability to mate
Males: (Number of males with confirmed mating/Total number of males cohabited) X 100
Females: (Number of sperm positive females/Total number of females cohabited) X 100

Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males: (Number of males impregnating a female/Total number of males with confirmed mating) X 100
Females: (Number of pregnant females/Number of sperm positive females) X 100

Gestation index: Measure of pregnancy that provides at least one live pup; (Number of females with live born pups/Number of pregnant females) X 100
Offspring viability indices:
Post-implantation mortality (intrauterine mortality): ((Number of implantations - Number of liveborns)/Number of implantations) X 100

Post-natal mortality: ((Number of liveborns - Number live pups on PND 13)/Number of liveborns) X 100

Survival Index : (Number of live pups on PND 13/Number of pups born) X 100

Sex ratio: ((Number of pups examined - Number of males/females)/Number of pups examined) X 100

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Alopecia on the right forelimb was observed on one female animal at 300 mg/kg bw/day (1/12) on gestation days 11-21 and for the same female alopecia on the forelimbs, thorax and abdomen was observed on lactation days 0-13. Piloerection was recorded for one female animal at 1000 mg/kg bw/day (1/12) on gestation day 23 probably due to the delivery and not related to the treatment. Because clinical signs of toxicity occurred at a low incidence and were not necessarily dose related, these effects were not considered treatment-related.

There were no clinical signs in male or female animals the weekly detailed clinical observations during the course of the recovery period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was observed at the higher mean body weight gain in male animals at 100 and 300 mg/kg bw/day between Days 41 and 48. In the male animals at 1000 mg/kg bw/day, the mean body weight gain exceeded the control value from Day 20 up to Day 48 except between Days 34 and 41 and for the study overall (between days 0 and 48).

In female animals at 300 mg/kg bw/day, statistical significance with respect to the control was observed at the higher mean body weight gain between Days 7 and 13 and between lactation days 0 and 4.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, slight but statistically significant difference with respect to the control was noted for the lower mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 1000 mg/kg bw/day.

In female animals, slightly but statistically higher values were observed in some parameters: in the mean concentration of hemoglobin (HGB) and mean hematocrit value (HCT) at 100 and 300 mg/kg bw/day, in the mean corpuscular (erythrocyte) volume (MCV) and mean corpuscular (erythrocyte) hemoglobin (MCH) at 300 mg/kg bw/day.

These differences with respect to control were judged to be toxicologically not relevant due to the minor degree or in the lack of dose relevance and values were within the historical control range (MCV, MCH) or were marginal changed (MCHC, HGB, HCT) to the historical control ranges.

The mean percentages of monocytes (MONO) and basophil granulocytes (BASO) were higher than in the control group in male animals at 1000 mg/kg bw/day at the end of the recovery period.

In the female animals at 1000 mg/kg bw/day, all examined parameters were comparable to the control at the end of the recovery period.

These differences with respect to control in male animals were judged to be indicative of biological variation and toxicologically not relevant. These differences were only observed at the end of the recovery period but not at the end of the treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to their control, the mean concentration of glucose (GLUC) was statistically significantly higher in male animals at 100, 300 and 1000 mg/kg bw/day. All individual values were well within the historical ranges and statistical significances were mainly originated from the relatively low value of the control group. Therefore, this was considered to have no toxicological relevance.

In female animals, slightly but statistically significantly higher mean concentration of sodium (Na+) was detected at 300 and 1000 mg/kg bw/day without any toxicological importance.

The mean activity of alanine aminotransferase (ALT) was higher than in the control group in male animals at 1000 mg/kg bw/day at the end of the recovery period. The slight difference with respect to control in ALT activity was judged to be indicative of biological variation and toxicologically not relevant in the lack of histopathological alterations and similar finding was not detected at the end of the treatment period.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In animals selected for full histological examinations, minimal alveolar emphysema in the lungs (1/5 control male, 1/5 male at 1000 mg/kg bw/day) was detected sporadically.

Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 control male; 1/5 male at 1000 mg/kg bw/day; 1/5 control female) was observed is some animal. Since the effect was observed in the control and high dose group. The observation was not judged to be treatment related.

Tissue formation (necrotic debris surrounded by fibrotic capsule) in the abdominal cavity was observed in one control male animal (1/6).

One female at 1000 mg/kg bw/day had a cyst in the ovary (one side).

The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all cases except one male animal (1/12 at 1000 mg/kg bw/day). This animal had smaller than normal seminal vesicle (one side) and histological examination revealed decreased amount of secrete in the seminal vesicle, without any other pathological lesion (inflammation, fibrosis etc.).
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no differences between the control and treated groups (100, 300 and 1000 mg/kg bw/day) in the number or percentage of animals with regular or irregular cycles, in the mean length of estrous cycle, in the mean number of days in pro-estrous, estrous or diestrus, in the number or percentage of animals in prolonged estrous or diestrous during the pre experimental and pre-mating periods.

The mean number of estrous cycle at 300 mg/kg bw/day was slightly but statistically significantly lower than in the control group during the pre-mating period. Similar finding was not detected at the high dose treated female animals, therefore this sight change had no toxicological relevance.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significances with respect to the relevant control were noted for the shorter mean pre-coital interval and conceiving days in female animals at 300 mg/kg bw/day. This was considered to have no toxicological relevance.

There were no differences between the control and test item treated groups in copulatory, fertility indices (male and female animals) and in gestation indices (females).

The mean duration of pregnancy was slightly longer than in the control group in dams at 100 and 1000 mg/kg bw/day.

At 1000 mg/kg bw/day, statistical significance was noted for the higher mean percentage of post-implantation loss and for the lower mean number of implantations with respect to their control.

There were no statistically significant differences between the control and test item treated groups in the number of pregnant females and dams delivered, in the mean number of total births, liveborns, stillborns, viable pups on post-natal day 0 or in the live birth indices.

The statistical significances at the mentioned parameters (post-implantation loss, number of implantation sites, duration of pregnancy) comparing to their control were mainly originated from the relative lower or higher mean values of control group with respect to the historical control. The values remained well close to the historical control. Therefore these changes were considered to be toxicologically not relevant.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Key result
Critical effects observed:
no
Reproductive function: sperm measures:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of offspring showing clinical signs (cold, not suckled, found dead, missing) was the lowest in the high dose group.

Occasionally, other clinical signs were also observed: cyanotic skin (1% at control; 1% at 100 mg/kg bw/day), smaller than normal pup (1% at 1000 mg/kg bw/day), cachectic pup (1% at 1000 mg/kg bw/day), alopecia (5% at 100 mg/kg bw/day), which however were considered to have no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was observed at the higher mean body weight gain in male animals at 100 and 300 mg/kg bw/day between Days 41 and 48. In the male animals at 1000 mg/kg bw/day, the mean body weight gain exceeded the control value from Day 20 up to Day 48 except between Days 34 and 41 and for the study overall (between days 0 and 48). In female animals at 300 mg/kg bw/day, statistical significance with respect to the control was ob served at the higher mean body weight gain between Days 7 and 13 and between lactation days 0 and 4.

Slightly higher mean body weight gain was observed in male animals at 1000 mg/kg bw/day comparing to their control during the first week of the recovery period. The mean body weight and body weight gain were similar to that of the control group in female animals at 1000 mg/kg bw/day during the course of post-treatment period.

These slight changes in the mean body weight gain of test item treated male animals during the treatment and recovery periods and in female animals at 300 mg/kg bw/day did not result in changes in the mean body weight therefore were considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically significant difference with respect to the control was noted for the slightly higher mean T4 thyroid hormone (free T4) level in PN13 offspring at 1000 mg/kg bw/day. This slight difference was considered to be toxicologically not relevant as all individual values were within or marginal to the historical control range.
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant difference with respect to the control was detected at the slightly higher mean number of male pups and at the slightly lower mean number of female pups at 100 mg/kg bw/day. The difference was of minor degree and the mean number of male and female pups per litter did not show statistical significance. Therefore, it was considered to be toxicologically not relevant.

The mean anogenital distance (absolute and normalized) was statistically significantly longer in male pups of the 1000 mg/kg bw/day treated dams. Only a shortened anogenital distance has an impact on the fertiliy of males and therefore the effect was not judged to be toxicologically relevant.

In female pups, statistical significance was observed at the longer mean absolute anogenital distance at 1000 mg/kg bw/day and at the slightly lower normalized anogenital distance at 300 mg/kg bw/day.

Due to the minor degree and the higher pup weights in the high dose, these slight differences with respect to the control were not considered to be toxicologically relevant.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day autolyzed visceral organs were detected in one pup (1/2) on post-natal day 2, and one dead pup (1/2) was cachectic with empty stomach and its visceral organs were partially autolyzed on post-natal day 13.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In an oral gavage study conducted according to OECD 422 and to GLP (reliability score 1) the NOAEL for butylmonoglycol sulphate, Na-salt relating to repeated dose (parental systemic) effects and to reproductive toxicity was at least 1000 mg/kg bw/day, as no significant adverse effects were observed in rats. No toxic effects were observed in offspring; therefore, the NOAEL for developmental toxicity was established at 1000 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészégügyi Intézet
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
other: Han:WIST of Wistar origin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 85-90 days; Females: 85-90 days
- Weight at study initiation: Males: 315-396 g; Females: 190-230 g
- Housing: Before mating: 2 animals of the same sex/cage; Mating: 1 male and 1 female/cage; Pregnant females: individually; Males after mating: 2 animals/cage; Recovery animals: 2 or 3 animals of the same sex/cage
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice provided ad libitum
- Water: tap water provided ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod: Artificial light, from 6 a.m. to 6 p.m

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Butylmonoglycol sulphate, Na-salt was formulated in the vehicle (distilled water) at concentrations of 20, 60 and 200 mg/mL in the formulation laboratory of the Test Facility daily.

VEHICLE
- Amount of vehicle: Control animals were handled in an identical manner to the test groups receiving vehicle (5 mL/kg bw).
- Lot/batch no. (if required): 1801-5512; 1801-5512; 1803-5501
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study.
Duration of treatment / exposure:
49 days (male animals) or 50-56 days (female animals); the day of first treatment is considered as Day 0 of examination.
Frequency of treatment:
7 days/week, every day at a similar time (±2 hours)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals per sex per dose
5 animals per sex for control and high dose group as recovery group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen by the Sponsor on the basis of the results of a preliminary dose range finding study with Butylmonoglycol sulphate, Na-salt in rats (Study no. 904-400-3039). The high and mid-high doses were chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Rationale for animal assignment: All male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups was as uniformly as practicable. Grouping was aided by SPSS/PC software, verifying the homogeneity and variability between the groups according to the actual body weight.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made on parental animals once a day, after the administration at approximately the same time. Detailed examinations were made at the times of wee kly weighing, prior to and during the mating and until necropsy.
- Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed examinations were made at the times of weekly weighing, prior to and duri ng the mating and until necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy (treatment and recovery periods). Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10. Body weight was measured on day of necropsy for female animals subjected to organ weighing. Animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment period.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Anaesthetic used for blood collection: Yes; Isofluran anesthesia
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table [No. 1 and 2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table [No. 3 and 4] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the last exposure week (Day 49 (corresponding to PND 9-12 for dams) before the blood sampling
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity

IMMUNOLOGY: No

OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; all animals were subjected to a full detailed gross necropsy. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes; the tissues indicated in Table 5 were prepared for microscopic examination and weighed, respectively.
Statistics:
The statistical evaluation of appropriate data (marked † above) was performed with the statistical program package SPSS PC+4.0.

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.

Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis an alysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi² test was performed if feasible.

For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Alopecia on the right forelimb was observed on one female animal at 300 mg/kg bw/day (1/12) on gestation days 11-21 and for the same female alopecia on the forelimbs, thorax and abdomen was observed on lactation days 0-13. Piloerection was recorded for one female animal at 1000 mg/kg bw/day (1/12) on gestation day 23 probably due to the delivery and not related to the treatment. Because clinical signs of toxicity occurred at a low incidence and were not necessarily dose related, these effects were not considered treatment-related.

There were no clinical signs in male or female animals the weekly detailed clinical observations during the course of the recovery period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was observed at the higher mean body weight gain in male animals at 100 and 300 mg/kg bw/day between Days 41 and 48. In the male animals at 1000 mg/kg bw/day, the mean body weight gain exceeded the control value from Day 20 up to Day 48 except between Days 34 and 41 and for the study overall (between days 0 and 48). In female animals at 300 mg/kg bw/day, statistical significance with respect to the control was ob served at the higher mean body weight gain between Days 7 and 13 and between lactation days 0 and 4.

Slightly higher mean body weight gain was observed in male animals at 1000 mg/kg bw/day comparing to their control during the first week of the recovery period. The mean body weight and body weight gain were similar to that of the control group in female animals at 1000 mg/kg bw/day during the course of post-treatment period.

These slight changes in the mean body weight gain of test item treated male animals during the treatment and recovery periods and in female animals at 300 mg/kg bw/day did not result in changes in the mean body weight therefore were considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, slight but statistically significant difference with respect to the control was noted for the lower mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 1000 mg/kg bw/day.

In female animals, slightly but statistically higher values were observed in some parameters: in the mean concentration of hemoglobin (HGB) and mean hematocrit value (HCT) at 100 and 300 mg/kg bw/day, in the mean corpuscular (erythrocyte) volume (MCV) and mean corpuscular (erythrocyte) hemoglobin (MCH) at 300 mg/kg bw/day.

These differences with respect to control were judged to be toxicologically not relevant due to the minor degree or in the lack of dose relevance and values were within the historical control range (MCV, MCH) or were marginal changed (MCHC, HGB, HCT) to the historical control ranges.

The mean percentages of monocytes (MONO) and basophil granulocytes (BASO) were higher than in the control group in male animals at 1000 mg/kg bw/day at the end of the recovery period.

In the female animals at 1000 mg/kg bw/day, all examined parameters were comparable to the control at the end of the recovery period.

These differences with respect to control in male animals were judged to be indicative of biological variation and toxicologically not relevant. These differences were only observed at the end of the recovery period but not at the end of the treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to their control, the mean concentration of glucose (GLUC) was statistically significantly higher in male animals at 100, 300 and 1000 mg/kg bw/day. All individual values were well within the historical ranges and statistical significances were mainly originated from the relatively low value of the control group. Therefore, this was considered to have no toxicological relevance.

In female animals, slightly but statistically significantly higher mean concentration of sodium (Na+) was detected at 300 and 1000 mg/kg bw/day without any toxicological importance.

Statistically significant difference with respect to the control was noted for the slightly higher mean T4 thyroid hormone (free T4) level in PN13 offspring at 1000 mg/kg bw/day. This slight difference was considered to be toxicologically not relevant as all individual values were within or marginal to the historical control range.

The mean activity of alanine aminotransferase (ALT) was higher than in the control group in male animals at 1000 mg/kg bw/day at the end of the recovery period. The slight difference with respect to control in ALT activity was judged to be indicative of biological variation and toxicologically not relevant in the lack of histopathological alterations and similar finding was not detected at the end of the treatment period.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was noted for the higher mean liver weights (absolute) in male animals at 1000 mg/kg bw/day.

In the female animals at 100 mg/kg bw/day, the mean weights of liver (absolute and relative to brain weight) and adrenal glands (absolute) were slightly lower than in the control group. The mean kidney weight relative to body weight was higher than in the control group.

At 300 mg/kg bw/day, statistical significance was observed at the lower mean weights of adrenal glands (absolute and relative to body and brain weights) in female animals.

At 1000 mg/kg bw/day, statistical significance was noted for the slightly higher mean fasted body weight and at the lower mean weights of adrenal glands (absolute and relative to body and brain weights) for females.

At the end of the recovery period, the mean brain weight relative to body weight was slightly but statistically significantly lower and the mean body weight relative to brain weight was statistically significantly higher compared to the control group in male animals at 1000 mg/kg bw/day.

In the female animals of the 1000 mg/kg bw/day recovery group, the weight of the examined organs was comparable to their controls.

There were no related pathological changes (clinical chemistry, histopathology) therefore these slight changes in the liver of male animals dosed with the high dose were considered to be toxicologically not relevant.

Changes in the organ weights of female animals were of minor degree and not related to doses (liver, kidneys, adrenal glands) thus were considered to be toxicologically not relevant.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group, yellow-brown formation (0.5 x 1 cm in size) in the abdominal cavity was observed in one male animal (1/12).

In male animals at 1000 mg/kg bw/day, smaller than normal seminal vesicle on the left side (1/12) and pyelectasia (1/12) were observed.

In female animals at 1000 mg/kg bw/day, interrupted left horn of the uterus with liquid content (1/12) was observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In animals selected for full histological examinations, minimal alveolar emphysema in the lungs (1/5 control male, 1/5 male at 1000 mg/kg bw/day) was detected sporadically.

Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 control male; 1/5 male at 1000 mg/kg bw/day; 1/5 control female) was observed is some animal. Since the effect was observed in the control and high dose group. The observation was not judged to be treatment related.

Tissue formation (necrotic debris surrounded by fibrotic capsule) in the abdominal cavity was observe d in one control male animal (1/6). One female at 1000 mg/kg bw/day had a cyst in the ovary (one side).

The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all cases except one male animal (1/12 at 1000 mg/kg bw/day). This animal had smaller than normal seminal vesicle (one side) and histological examination revealed decreased amount of secrete in the seminal vesicle, without any other pathological lesion (inflammation, fibrosis etc.).
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an oral gavage study conducted to OECD 422 and according to GLP (reliability score 1) the NOAEL for butylmonoglycol sulphate, Na-salt relating to repeated dose (parental systemic) toxicity was at least 1000 mg/kg bw/day, as no significant adverse effects were observed in rats during the study period.