Magnesium octadecylbenzenesulphonate

Toxicity to reproduction

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08/03/2018 to 21/01/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

Batch No.: E00873-025-001
Physical Description: Black solid
Storage Conditions: Kept in a controlled room temperature area

Specific details on test material used for the study:

Identification: EXP1313100
Batch (Lot) No.: E00873-025-001/E00873-085
Expiration Date: 31 Dec 2019
Physical Description: Solid
Purity: 100%
Storage Conditions: Ambient temperature, protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Doses were administered orally by gavage at a dose volume of 10 mL/kg (Day 1 to 17 of dosing) or 5 mL/kg (Day 18 of dosing onwards) due to transient clinical observations of abnormal gait and subdued behaviour being noted for all animals, including controls. It was decided after consultation with the Sponsor, that the dose volume would be lowered to 5 mL/kg from Study Day 18 onwards, in order to lower the overall volume of the vehicle propylene glycol being administered to each animal

Details on mating procedure:

Pairing was on a 1 male to 1 female basis. During the evening (after 16:00 hours) of Day 15 of dosing, females were housed with their allocated co-group male partner. The animals were paired in ascending numerical order.

Vaginal lavages were taken early each morning from the day of pairing until mating had occurred and the stage of oestrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated as Day 0 of gestation. The pairing period for each pair of animals was a maximum of 12 nights.

For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (oestruses passed without a sign of mating) was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses described below were performed by GC with Flame Ionisation Detection (FID) using a validated analytical procedure

Duplicate sets of top, middle and bottom samples (duplicate middle only for control) for each sampling time point were sent to the analytical laboratory. Triplicate sets of top, middle and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples. Samples were 1 mL for Group 1, 0.5 mL for Group 2, and 0.1 mL for Groups 3 and 4, which were collected into appropriately sized volumetric flasks before being stored as required protected from light, either in a refrigerator set to maintain 4°C or at ambient temperature. Concentration results were acceptable when mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable when it was within or equal to ± 20%. Homogeneity results were considered acceptable when the relative standard deviation (RSD) of the mean value at each sampling location was <= 10% for each group. After acceptance of the analytical results, backup samples were discarded.

All analytical results were acceptable and within acceptance criteria.

Duration of treatment / exposure:

The males were dosed once daily for at least 4 weeks overall, starting from 2 weeks prior to mating (i.e. sacrificed on Day 30).

The females were dosed once daily from 2 weeks prior to mating, then continuing until the day prior to termination (at least Day 13 of lactation).

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

The oral route of administration was selected for this study as this route has been defined by the Sponsor as a possible route of human exposure during manufacture, handling or use. The oral route was considered worst case and the data will provide part of a rational basis for toxicological risk assessment in man.

Dose levels were chosen following review of data from a 28 day rat study (Project 505705) performed by the Sponsor, in an attempt to produce graded responses to the test item. The high dose level was chosen to produce some toxic effects, but not excessive lethality that would prevent meaningful interpretation. The mid and low dose levels were chosen to explore the dose range.

Examinations

Parental animals: Observations and examinations:

Animals were observed twice daily, once at the start and once towards the end of the working day throughout the study, for general health/mortality and moribundity.

Animals were removed from the cage daily from Week -1 for detailed examination.

Animals were removed from the cage daily from Week -1 for detailed examination.

Animals were individually weighed weekly during pretreatment and then males daily during the dosing period. Females were weighed daily up to Day 42 of dosing and thereafter periodically during lactation. Weights have been presented weekly for males and for females during the pre-mating period and for females on Days 0, 4, 7, 11, 14, 17 and 20 of gestation and Days 1, 4, 7, 10 and 13 of lactation.

Food consumption was quantitatively measured weekly for both sexes until pairing for mating starting Week -1. For mated females, food consumption was recorded over the periods Day 0-7, 7-14 and 14-20 of gestation, and Days 0-7 and 7-13 of lactation.

Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

Oestrous cyclicity (parental animals):

Vaginal lavages were taken early each morning and the stages of oestrous observed were recorded over a 2 week period prior to test item administration (including spares), then continuing through pre-mating test item administration and the mating period.

Vaginal smears were not examined on the morning of necropsy to determine the stage of oestrus cycle to allow correlation with histopathology of the ovaries, this was a deviation in the protocol. Instead, histopathological examination of the vagina were conducted.

Sperm parameters (parental animals):

For all Group 1 and Group 4 males, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 5 micrometers, and stained with PAS/haematoxylin.

Litter observations:

The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups were born) was designated as Day 0 of lactation. The duration of gestation in days was calculated.

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined daily for the presence of milk in the stomach and for any externally visible abnormalities. Individual pup weights were taken from each litter on Day 1 of lactation, and each litter was weighed en masse (by sex) on days 4, 7, 10 and 13 of lactation. Pups were individually identified via toe-marking (details have been recorded in the study records).

On Day 4 of lactation, litter size was standardised to 8 pups per litter, where possible 4 males and 4 females, by culling of extra pups via random selection. Extra pups were used for blood sampling and/or necropsy procedures or killed without necropsy as per Test Facility SOPs. Where the total number of pups in a litter on Day 4 of lactation was =8 pups, no litter size adjustment occurred.

Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Any externally abnormal decedent pup was preserved; externally normal pups were discarded.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest. This was replaced when it had become soiled.

Assessment of ano-genital distance was measured for all pups in each litter on PND1. Nipple retention was assessed for males on PND13. On the day of scheduled necropsy (i.e. Day 14-16 of lactation), the external genitalia of all pups were examined by assessment of preputial separation or vaginal opening to determine pre-weaning physical development. It was noted whether genitalia development was normal or abnormal.

Postmortem examinations (parental animals):

Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female. The uteri of the non-pregnant female was stained using 10% aq (v/v) ammonium sulphide solution and examined for implantation sites.

Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

The organs identified below were weighed at necropsy for all adult animals. Paired organs were weighed together. Organs were weighed before fixation unless otherwise noted. Organ to body weight ratios (using the terminal body weight) were calculated.

Brain
Epididymis a
Gland, adrenal a
Gland, pituitary
Gland, prostate
Gland, thyroida b
Heart
Kidney a
Liver
Lung
Ovary a
Spleen
Testis a
Thymus
Uterus
a = Paired organ weight.
b = Organ weighed after fixation.

At necropsy between Days 14 to 16 of lactation, thyroid/parathyroid gland was fixed and later weighed for one male and one female pup per litter

Representative samples of the tissues identified below were collected from all adult animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Animal identification
Artery, aorta
Bone marrow, femur
Bone marrow, sternum
Bone, femur
Bone, sternum
Bone, rib
Brain
Cervix
Epididymis
Eye a
Gland, adrenal
Gland, harderian
Gland, lacrimal
Gland, mammary
Gland, parathyroid
Gland, pituitary
Gland, prostate
Gland, salivary, mandibular
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue (Peyer’s Patches)
Heart
Kidney
Large intestine, caecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node, mandibular
Lymph node, mesenteric
Muscle, skeletal
Nasal cavity
Nerve, optic a
Nerve, sciatic
Oesophagus
Ovary
Oviduct
Pancreas
Pharynx
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testisb
Thymus
Tongue
Trachea
Ureter
Urinary bladder
Uterus
Vagina
a = Preserved in Davidson’s fixative.
b = Preserved in Modified Davidson’s fixative.

Histology - The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin:
• Testes of all Group 1 and Group 4 males, examined for staging of spermatogenesis (qualitative evaluation)
• Group 1 and 4 female ovaries
• All Group 1 and 4 animals – liver
• Adrenal glands of all animals (all groups)
• Gross lesions of all animals (all groups)
• Vagina (all adult females) - to determine the stage of oestrus cycle and allow correlation with histopathology of ovaries.

In addition for all Group 1 and Group 4 males, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 5 micrometers, and stained with PAS/haematoxylin.

The tissues listed above were subjected to histopathological evaluation by a veterinary pathologist with training and experience in laboratory animal pathology.

Postmortem examinations (offspring):

Necropsy (Offspring Found Dead or Euthanised Prematurely) - Where practicable, animals found dead or killed were sexed, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, but were not examined. Externally normal pups were discarded.

Necropsy (Offspring at Scheduled Termination) - These animals were examined for gross lesions. Samples of any abnormal tissues were taken and fixed in neutral buffered 10% formalin. All pups that had thyroid samples taken and remaining pups from specified litters were examined by open dissection for internal sex confirmation, see protocol deviations and other events in Appendix 1. Thyroid with parathyroid were collected from one pup of each sex per litter, see protocol deviations and other events in Appendix 1. The remaining carcasses were discarded.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 0.1%, 1%, and 5% levels.

Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to sex and occasion. Descriptive statistics (number, mean and standard deviation or %CV or SE when deemed appropriate) have been reported whenever possible. Values are also expressed as a percentage of control values when deemed appropriate. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric/Non-Parametric

Levene’s test was used to assess the homogeneity of group variances.
Datasets with at least 3 groups were compared using an overall one-way ANOVA F test when Levene’s test was not significant or the Kruskal-Wallis test when it was. When the overall F test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Reproductive indices:

For each group:

Fertility Index (male) = Number siring a litte/Number paired

Fertility Index (female) = Number pregnant/Number paired

Gestation Index = Number bearing live pups/Number pregnant

Offspring viability indices:

For each litter and group:

Birth Index = Total number of pups born (live and dead)/Number of implantation scars

Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)

Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

From Day 16 up to Day 41 of dosing, abnormal gait and/or subdued behaviour were noted for all animals, including controls. However, these observations were concluded to be due to the vehicle propylene glycol and not EXP1313100-related. Ploughing was noted for all or the majority of animals per group, including contols (observations of salivation were also seen for females) from Day 10 up to Day 55 of dosing. The clinical observations (abnormal gait and/or subdued behaviour, ploughing) and reduced food consumption noted for all animals, including controls, were considered to be due to the vehicle propylene glycol and not EXP1313100-related. Research has shown that oral exposure to high doses of propylene glycol causes CNS depression, the clinical signs of which in rats include ataxia and decreased spontaneous motor activity

Ploughing and salivation were also considered to be due to the vehicle propylene glycol as they were observed at the same incidence and over the same time period in control and test item treated animals. Chewing action was noted for 2 females given 300 or 450 mg/kg/day and due to the low incidence but in test item groups only, could not be solely attributed to the vehicle propylene glycol.

The other clinical observations noted were only observed in a few animals in a group (including controls) or were of the type normally observed in pregnant rats and therefore were considered not to be EXP1313100-related.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the course of this study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males, there were no test item-related effects on body weight or body weight gain over the 5 week dosing period. Females at all EXP1313100 dose levels appeared to have a slightly higher weight gain (19% to 36%) over the 15 days prior to mating, but after the differences in group mean weights on Day 0 of gestation were taken into account, this was considered not to be test item-related. Also, there was no effect on female body weight gain during gestation or lactation, compared with controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During Weeks 1 and 2 of dosing, food consumption was decreased by up to 33% for controls and all EXP1313100 groups of both sexes, compared to the relevant group values during Week -1. However, this was concluded to be due to the vehicle propylene glycol and not EXP1313100-related

Females during gestation (controls and test item treated groups) ate more than pre-mating, although generally values were similar to those recorded during Week -1 and slightly lower than would normally be expected. During lactation, food consumption increased above previous intake, and was generally similar for controls and test item treated groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dose level-related slightly higher values of liver enzymes were noted at up to 450 mg/kg/day EXP1313100. For both sexes at all dose levels, ALT was higher by 1.2 or 3.4-fold, for males at 300 or 450 mg/kg/day ALP and CK values were higher than controls by up to 1.3 or 2.0-fold respectively, and for females CK was higher by up to 1.7-fold at all EXP1313100 dose levels. For females at 450 mg/kg/day only, AST was slightly higher by 1.2-fold and cholesterol was slightly lower by 0.8-fold.

There were no test item-related effects on any other clinical chemistry parameters. All values were considered to be within normal biological variation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic findings.

One male (Animal 3002M) at 300 mg/kg had granulomatous inflammation in the lungs, which was considered to be secondary to a gavage accident.

Other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

Other effects:

no effects observed

Description (incidence and severity):

There were no biologically relevant differences in total T4 levels noted among the different groups of adult males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no EXP1313100-related effects noted for oestrus cycles during the dosing period.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no EXP1313100-related effects on mating performance, fertility and gestation indices or duration of gestation at any dose level. One male and female given 100 mg/kg/day (Animals 2007M and 5503F) failed to mate and another female given 300 mg/kg/day (Animal 3503F) failed to produce a live litter but due to the low incidence of these findings and lack of similar findings at the highest dose level, they were considered to be incidental.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The litter weight of test item treated groups was slightly lower than controls by up to 18% on Day 4 of lactation, as a consequence of the slightly lower number of pups born, compared with controls. However, the number of control pups born per litter was considered to be on the higher side of the normal range.

EXP1313100 litter and pup weight were comparable with controls after litter size was standardised to 8 pups per litter but thereafter, pup weight gain at 100 or 450 mg/kg/day was not as great as that of control pups and on Day 13 of lactation, the litter weight for these groups was 8% lower. However, in the absence of dose-related response, there being no effect on litter or pup weight at 300 mg/kg/day on Day 13 of lactation, this change could not be confirmed as a test item-related effect.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no EXP1313100-related effects noted

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no EXP1313100-related effects noted on PND1 for ano-genital distance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipple retention for male pups on PND13 was normal and there were no abnormalities for external genitalia of any pup (assessed by means of preputial separation or vaginal opening) on Day 14-16 of lactation.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no EXP1313100-related effects noted

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no biologically relevant differences in total T4 levels noted among the different groups of male PND13 pups nor different groups of female PND13 pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of EXP1313100 by once daily oral gavage to Crl:CD(SD) Sprague Dawley rats (males for 4 weeks starting from 2 weeks prior to mating, and females from 2 weeks prior to mating continuing until at least Day 13 of lactation) was well tolerated at dose levels up to 450 mg/kg/day. Dose level-related slightly higher levels of liver enzymes were noted at up to 450 mg/kg/day, and cholesterol was slightly lower for females at 450 mg/kg/day. There were no effects on reproduction and development that were considered to be related to the administration of EXP1313100.

Executive summary:

The objective of this study wastoassess the toxicity of EXP1313100in the rat after oral gavage administration(males for 4 weeks starting from 2 weeks prior to mating, and females from 2 weeks prior to mating continuing until at least Day 13 of lactation)and to provide initial information on possible effects on reproduction and development.

The following parameters and end points were evaluated in this study: clinical observations, body weights, body weight gains, food consumption, mating performance and fertility indices, duration of gestation and overall litter performance, litter survival indices, litter and pup weights, clinical pathology parameters (clinical chemistry, Thyroxine [T4]), gross necropsy findings, organ weights, organ weights relative to body weights and histopathologic examinations.

The clinical observations (abnormal gait and/or subdued behaviour, ploughing) and reduced food consumption noted for all animals, including controls, were considered to be due to the vehicle propylene glycol and not EXP1313100-related.

There were dose level-related slightly higher values of liver enzymes,ALT and CK (for both sexes), ALP and AST (for males or females, respectively)at up to 450 mg/kg/day and also cholesterol was slightly lower for females, but at 450 mg/kg/day only.

 

For all other parameters, there were no toxicologically significant changes considered to be related to administration ofEXP1313100.

In conclusion, administration of EXP1313100by once daily oral gavage to Crl:CD(SD) Sprague Dawley rats (males for 4 weeks starting from 2 weeks prior to mating, and females from 2 weeks prior to mating continuing until at least Day 13 of lactation) was well tolerated at dose levels up to 450 mg/kg/day. Dose level-related slightly higher levels of liver enzymes were noted at up to 450 mg/kg/day, and cholesterol was slightly lower for females at 450 mg/kg/day. There were noeffects on reproduction and development that were considered to be related to the administration of EXP1313100.