Sodium (xylenes and 4-ethylbenzene)sulfonates

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Based on Article 41 of Regulation (EC) No 1907/2006 (the REACH Regulation), ECHA requests you to submit information on:
Extended one-generation reproductive toxicity study
(Annex X, Section 8.7.3.; test method OECD TG 443) in rats, oral route with the registered substance specified as follows:
-Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity); and
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium (xylenes and 4-ethylbenzene) sulfonates

EC Number:

701-037-1

Cas Number:

1300-72-7

Molecular formula:

-

IUPAC Name:

Sodium (xylenes and 4-ethylbenzene) sulfonates

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118

Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species and comparability with the studies already carried out

Sex: males and females

Age of animals: 5 weeks at arrival

Acclimatization: 5 days

Number of animals: 28 females and 28 males per dose group + 2 males and 2 females for control of microbiological background (totally 228 animals)

Housing conditions: IVC – acclimatization of P animals, 10 week pre-mating period (due to reconstruction of SPF animal house)
SPF – from mating till the end of study

Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother

Food: complete pelleted low-phytoestrogen diet for rats and mice in SPF breeding (with certificate): Altromin 1324 phytoestrogen-deficient, Batch No. 202106290607, 202108311308

Water: drinking water ad libitum, Regulation No. 252/2004 Czech Coll. Of Law, Ministry of Health

Light cycle: 12 hour light / 12 hour dark

Microclimate: 19-25 °C, relative- humidity 30-70%

Bedding: sterilized soft wood fibers Lignocel (producer: JRS GMBH+Co.KG, Rosenberg, Germany)
Selection of animals: random selection according to the internal SOP No.42 – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the group mean weight

Identification of animals: the parental animals were identified by the colour marks on their fur, each cage was marked with the number of animals, sex, number of cage, name and dose level of the test item; pups were tattooed

Animal housing
The study was performed in IVC (Individually ventilated caging systems) during the acclimatization and pre-mating period (due to reconstruction of SPF animal rooms) according to internal SOP No. 217. Before the mating (till the end of study) the animals were housed in a SPF (Specified Pathogen Free) animal house of CETA in SPF conditions according to internal SOP No. 12. Animals were housed in the animal room, 2 rats of the same sex in one plastic cage (polycarbonate cages for rats TECNIPLAST, type 1291H, Italy)

Bedding
Sterilized shawings or Lignocel (Raw material: spruce; Producer: J. Rettenmaier & Söhne, Germany) were used.

Housing conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be: approximately 15 air changes per hour, a temperature of 22+3°C, a relative humidity of 30-70% and 12-hour light/12-hour dark cycle (according to internal SOP No. 46).

Feeding
Free access to food (ad libitum).

Diet
Altromin 1324 phytoestrogen-deficient
Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
Batch No: 202106290607, 202108311308

Water
Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Ministry of Health.

Additional information
The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriological contaminants every 2 months on a regular basis. Results are retained in the CETA archives. Certificates of drinking water analysis (performed twice a year) are retained in the CETA archives. Analysis of diet and water did not reveal any findings that could affect study integrity.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

According to ECHA request, the length of premating exposure was ten weeks to cover full spermatogenesis and folliculogenesis before mating.

P generation dosing
Males
10 weeks (pre-mating) → mating → 5 weeks post-mating (15 weeks)
Females – mothers
10 weeks (pre-mating) → max. 2 weeks mating (with vaginal smear – presence of sperm) → pregnancy (ca. 21 days) → lactation (depends on mating duration – minimum 16 weeks and maximum 18 weeks of application)
During the last week of pregnancy period (from the day 14 of pregnancy), the application volume of the test item suspension was adjusted in pregnant females every two days.

F1 generation dosing
At weaning (at PND 21) the animals were divided into the cohorts.
The oral administration of the F1 animals by the test item suspension started after weaning (from PND 22). The application volume of the test item suspension was adjusted every two days during the two weeks following weaning; then weakly.

Dosing of F1 generation
Males + Females
1A cohort: 10 weeks after weaning (weaning on PND21)
1B cohort: 11 weeks after weaning
1extra cohort: after weaning till attaining of puberty (see chapter Balano-preputial separation or vaginal patency).

Frequency of treatment:

Once daily

Details on study schedule:

The test item will be administered to the stomach by gavage as suspension in vehicle. The test item concentration at single dose level will be adjusted so that the administered volume will be constant at all dose levels – 1 ml/100 g body weight.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

108 mg/kg bw/day (nominal)

Remarks:

equivalent to 100 mg/kg/day a.i.

Doses / concentrations 3

Dose / conc.:

323 mg/kg bw/day (nominal)

Remarks:

equivalent to 300 mg/kg/day a.i.

Doses / concentrations 4

Dose / conc.:

1 076 mg/kg bw/day (nominal)

Remarks:

equivalent to 1000 mg/kg/day a.i.

No. of animals per sex per dose:

P generation
1. control – vehicle: 28 females and 28 males → at least 20 pregnant females
2. 100 mg/kg/bw day of a.i.: 28 females and 28 males → at least 20 pregnant females
3. 300 mg/kg/bw day of a.i.: 28 females and 28 males → at least 20 pregnant females
4. 1000 mg/kg/bw day of a.i.: 28 females and 28 males → at least 20 pregnant females

F1 generation (reproduction cohorts 1A and 1B + cohort 1ex)
1. control – vehicle: 20 males + 20 females
2. 100 mg/kg/bw day of a.i.: 20 males + 20 females
3. 300 mg/kg/bw day of a.i.: 20 males + 20 females
4. 1000 mg/kg/bw day of a.i.: 20 males + 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

Preparation of Experimental Animals
During the acclimatisation period the health condition of all animals was controlled daily. Then the animals were randomly divided into control and test groups and they were marked individually. Each rat of P generation was uniquely identified by veterinary hair dye - system of marks on the fur according to internal SOP 26 – Identification of animals. Pups (animals of F1 generations) were uniquely identified within litters using tattoos on PND 4. In animals divided into the cohorts, identification by veterinary hair dye was used.

Parental generation (P)
112 males and 112 females Wistar rats, approximately 5 weeks of age at arrival, were acclimatised for 5 days before initiation of dosing. The study included four groups of parental animals – 3 treated groups (100, 300 and 1000 mg of a.i./kg/day) and 1 control group (aqua pro iniectione only). Each group in the parental (P) generation consisted of 28 males and 28 females at the beginning of the study.

Mating Procedure
Parental males and females were mated after 10 weeks of administration: each female of P generation was placed with a single male of P generation from the same dose level (1:1 mating) until copulation occurs or 2 weeks had elapsed. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

First generation (F1)
At weaning (at PND 21) the animals were divided into the cohorts. The selected F1 pups were randomly assigned to cohorts of animals, as follows:

Cohort 1A = one male and one female/litter/group (up to 20/sex/group) serve for primary assessment of effect upon reproductive system and of general toxicity.

Cohort 1B = one male and one female/litter/group (up to 20/sex/group) would serve for follow-up assessment of reproductive performance by mating F1 animals. Cohort 1ex: an extra group of animals created only for evaluation of sexual maturation.

An extra group of pups was evaluated (Cohort 1ex): one male and one female/litter/group (up to 20/sex/group) serve only for evaluation of sexual maturation of F1 animals (see chapter Balano-preputial separation or vaginal patency).

In this study mating of F1 generation was not performed; the cohort 1B would serve only for obtaining additional histopathological data when results from cohort 1A would be equivocal.
Because there were not enough pups and/or sex of the pups in some litters; the inclusion of animals into a group 1A (primary general toxicity assessment) was preferred. Administration of the animals by the test item started at PND22 (post-natal day) and lasted till the necropsy day.

Surplus pups: Pups not selected into the cohorts (surplus pups) were euthanized and submitted for gross necropsy. The weight of organs and thyroid hormones was evaluated in selected surplus pups.

Necropsy: animals of Cohort 1A were necropsied after 10 weeks of application (approximate age at necropsy was 13 weeks). Animals of Cohort 1B were necropsied after 11 weeks of application (approximate age at necropsy was 14 weeks). Animals of Cohort 1ex were necropsied after attaining puberty.

Examinations

Parental animals: Observations and examinations:

P animals – IN-LIFE
mortality/viability: daily
health condition control: daily (before administration)
clinical observations: daily (after administration)
detailed clinical observation: weekly (when animals were weighed)
body weight: males – the first day of administration and then weekly;
females - the first day of administration and then weekly,
during pregnancy: 0., 7th, 14th, 20th day,
during lactation: 1st, 4th, 7th, 14th, 21st day;
food consumption: during premating period – weekly, during pregnancy and lactation – on the same days as body weight (except the mating period)

Oestrous cyclicity (parental animals):

Oestrous cycle stage: P-females: by cytology at termination

Sperm parameters (parental animals):

In P and F1-1A males of all groups surviving to scheduled necropsy the sperm parameters (motility, morphology, viability, count) were examined.
Sperm motility was evaluated immediately after sacrifice in all males (P + F1-1A) subjectively using a microscope. For the evaluation of sperm morphology, an epididymal sperm sample was examined as fixed preparations and 200 spermatozoa per sample were evaluated. The morphology was generated for control and high-dose P and F1-1A animals only (lower doses were not evaluated because the exposure-related effects were not recorded).
The sperm viability was evaluated by flow cytometry and sperm count was evaluated by using counting-chamber in 10 P and F1-1A males per group.

Litter observations:

F1 animals: up to weaning – IN-LIFE
mortality/viability: daily
health condition control: daily
clinical observations: daily
body weight: PND 1 – weight of whole litter
PND 4, 7, 14 and 21 – individual weight of pups
after weaning – every two days for two weeks, then weekly
litter standardisation: at PND 4
identification (tattoo) of pups: at PND 4
litter examination: when animals are weighed – at PND1: litter as a whole PND 4, 7, 14 and 21 – pups individually
anogenital distance: PND 4 (in pups of standardized litter only)
presence of nipples: PND 13

Postmortem examinations (parental animals):

P animals - TERMINAL
body weight: at termination
macroscopic examination: at termination
histopathology: post termination (in all high dose and controls)
oestrous cycle stage: P-females: by cytology at termination
sperm parameters: P-males: at or post termination
clinical biochemistry: 10 animals/sex/dose group at termination
thyroid hormones: 10 animals/sex/dose group at termination
haematology: 10 animals/sex/dose group at termination

Postmortem examinations (offspring):

F1 animals: post weaning – TERMINAL, all cohorts
body weight: at termination
gross necropsy: at termination
oestrous cycle stage: at termination

F1 animals – TERMINAL: specific to cohort 1
organ weight: at termination (specified in overview of examination)
histopathology: at termination (specified in overview of examination)
sperm parameters: at or post termination
clinical biochemistry: 10 animals/sex/dose group at termination
thyroid hormones: 10 animals/sex/dose group at termination
haematology: 10 animals/sex/dose group at termination
urinalysis: 10 animals/sex/dose group at termination
assessment of immunotoxicity: 10 animals/sex/dose group at termination

Statistics:

The software Statgraphic®Centurion (version XVII, USA) was used for statistical evaluation. Males/females from control group were compared with males/females from three treated groups. The results statistically significant on probability level 0.05 were indicated in the summary tables

Reproductive indices:

Male mating index
Female mating index
Male fertility index
Female fertility index
Gestation index

Offspring viability indices:

Viability index on PND4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

MALES: In treated males of all dose levels no clinical changes in health condition were recorded during the check-in and acclimatisation period, treatment-related effects were not detected during the health condition control and clinical observation in application period.

FEMALES: In treated females of all dose levels no changes in clinical condition were recorded during the check-in and acclimatisation period.
No clinical signs of toxicity related to the test item administration were recorded during the whole application period.
One exception was reported. Apathy, piloerection, white colour of ears and eyes were recorded in females No. 311 (the dose level 1000 mg/kg/day) after delivery. At 3 days postpartum, the female's health improved and no findings were observed. These changes were related to the difficult delivery and not related to the test item administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

MALES: Male No.62 (dose level 300 mg/kg/day) was found dead on 87 day of study (after 86 application). The exact cause of death could not be determined due to partial autolysis of organs. This death was considered accidental, unrelated to the administration of the test item
FEMALES: No females died during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

MALES: The body weight of treated group of males was comparable with the body weight of control males during the whole application period.
The statistic evaluation of body weight at 15th week and the necropsy weight did not show significant changes. The mean body weight increments of treated males of all dose levels were similar with control for the whole time of application period.

FEMALES:
- Before mating period: The mean body weight and body weight increments of control females and treated females at all dose levels were comparable in pre-mating period.

- Pregnancy: Females without parturition (non-pregnant) were not included in the evaluation of mean body weight increments during pregnancy. The mean body weight of treated mothers at the highest dose level (1000 mg/kg/day) and control mothers was similar. Statistically significantly decreased mean body weight in comparison with the control females was recorded in females at the dose levels 100 and 300 mg/kg/day at day 14 and 21 of pregnancy. It corresponds to the lower number of pups in these dose levels.

- Lactation: Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The mean body weight of treated mothers at the highest dose level (1000 mg/kg/day) and control mothers was similar. Statistically significantly decreased mean body weight in comparison with the control females was recorded in females at the dose levels 100 and 300 mg/kg/day during the whole lactation period. Statistically significant decrease was recorded on day 4, 7, 14 and 21 of lactation period in females at the dose levels 100 and 300 mg/kg/day.

- Necropsy body weight: No significant changes in necropsy body weight was recorded in treated females compared to control females

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MALES: The mean food consumption of treated males at all dose levels was similar to the food consumption of the control males.

FEMALES
- Pre-mating period: The mean food consumption of females at all dose levels was balanced with control females for the whole time of pre-mating period.

- Pregnancy: Females without parturition (non-pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy
The mean food consumption of mothers at all dose levels was well balanced with control mothers for the whole time of pregnancy.

- Lactation: Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period. The mean food consumption of treated mothers at all dose level was similar to control mothers.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

MALES: Fasted blood samples from a defined site were taken from first 10 adult males per dose at termination. The selected haematological parameters were examined. Statistically significantly increased value of RBC and decreased value of FIB were recorded in males at the dose levels 300 mg/kg/day. These changes were incidental (not related to the test item administration). Shortened (statistically significantly) Activated Partial Thromboplastin Time (APTT) was recorded in dosed males at all dose groups compared to control group of males. All values were in a historical control range.

FEMALES: Fasted blood samples from a defined site were taken from first 10 parturient females per dose group at termination. The selected haematological parameters were examined. Statistically significantly increased value of APTT was recorded in females at the all dose (without dose dependence).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

MALES: Fasted blood samples from a defined site were taken from first 10 adult males per dose at termination. The selected biochemical parameters were examined.
Statistically significantly changed values in treated group of males (compared to control group) were not detected.

FEMALES: Fasted blood samples from a defined site were taken from first 10 parturient females per dose at termination. The selected biochemical parameters were examined. Statistically significantly decreased concentration of creatinine was recorded at the dose levels 100 and 300 mg/kg/day.

Endocrine findings:

no effects observed

Description (incidence and severity):

MALES: Thyroid hormones (T4 + TSH)
Blood (serum) samples from first 10 adult males were assessed for thyroid hormones (T4 total and TSH).
No statistically significant differences in T4 and TSH concentration were noted between the treatment groups and the control group.

FEMALES: Thyroid hormones (T4 + TSH)
Blood (serum) samples from first 10 parturient females were assessed for thyroid hormones (T4 total and TSH).
No statistically significant differences in T4 and TSH concentration were noted between the treatment groups and the control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

MALES: Urinalysis was performed in first 10 males per group during the last week of the study. Statistical evaluation was performed for pH and volume of urine.
Statistically significantly decreased volume and increased pH value of urine were detected in males at the dose level 1000 mg/kg/day.
The presence of leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item. The presence of protein and blood in treated males is considered as accidental – common finding, not related to the test item administration (corresponds to the age of animals).

FEMALES: Urinalysis was performed in 10 females per group during the last week of the study. Statistical evaluation was performed for pH and volume of urine. Statistically significant differences in pH value and volume in treated females in comparison with the control females were not recorded.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

MALES: The behaviour, clinical status and activity of males in the dose groups 100, 300 and 1000 mg/kg/day were similar during the study and not different from males of the control group.
FEMALES: The behaviour, clinical status and activity of females in the dose groups 100, 300 and 1000 mg/kg/day were similar during the study and not different from females of the control group.
One exception was reported. Apathy, piloerection, white colour of ears and eyes were recorded in females No. 311 (the dose level 1000 mg/kg/day) after delivery. At 3 days postpartum, the female's health improved and no findings were observed. These changes were related to the difficult delivery and not related to the test item administration

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

MALES: No treatment-related changes were found in:
- Male genital tract (testes, epididymides, prostate gland)
- Liver
- Kidneys
- Pituitary gland
- Hemopoietic organs

FEMALES: No treatment-related changes were found in:
- Female genital tract (uterus, vagina, ovaries, mammary gland)
- Liver
- Kidneys
- Pituitary gland
- Hemopoietic organs

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The oestrous cyclicity of all parental females (28 females per dose) were examined by vaginal cytology during the 7th and 8th week of application.
No irregular cyclicity was recorded in females of control group and group 1000 mg/kg/day.
One females – No.237 (the dose level 100 mg/kg/day) had an irregular oestrous cycle. However this female became mother.
Females No. 262 and No.277 (the dose level 300 mg/kg/day), had an irregular oestrous cycle. Both females did not become mother.
All other females had a regular cycle (4-5 days).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

All survived males were examined.
Sperm motility was evaluated subjectively in all males (28 males per dose). A comparison of sperm motility in the control males and males from treated groups did not show differences.
Sperm morphology was evaluated in all (28 males per dose) males of all dose levels. Treatment related changes were not recorded.
Sperm count (caudal epididymis) was counted in first 10 males per dose and recalculated with respect to dilution and epididymal fluids weight. Administration of the test item did not affect the sperm count in the dosed group of males.
Sperm viability was evaluated in first 10 males per group. Decreased sperm viability was recorded in males at the dose levels 300 and 1000 mg/kg/day. At the dose level 1000 mg/kg, the viability of the sperms was decreased statistically significantly. However the male rats’ ability to produce sperm that can fertilise eggs was maintained.
On the other hand, because the viability of all treated groups of F1-males (after 10 weeks application) was the same and even better then viability of control group, histological examination did not reveal pathological finding and the spermatogenesis was not affected by the test item administration, the decreasing of viability in P-males was considered non- adverse effect of the test item.

Reproductive performance:

no effects observed

Description (incidence and severity):

The incidence of findings is expressed in numeric form and ranged in sequence of the dose levels 0-100-300-1000 mg/kg/day.

The number of females who mated was 28-28-28-28.
The number males who mated was 28-28-28-28.

The 28-27-26-26 females showed evidence of copulation and 24-21-20-21 females achieved pregnancy. The number of females with live pups born was 24-21-20-21.

The duration of mating (mean) was 2.61-3.22-1.96-4.04 days.
The duration of pregnancy (mean) was 21.33-22.00-21.75-21.48 days.

The number (mean) of implantation was 17.25-13.52-14.60-16.76.
The number (mean) of post-implantation loss was 2.75-1.48-2.10-2.57.
The number (mean) of post-natal loss was 0.13-0.52-0.10-0.81.

Results of reproductive indices:
Male mating index: 100.0-96.4-92.9-92.9
Female mating index: 100.0-96.4-92.9-92.9
Male Fertility index: 85.7-75.0-71.4-75.0
Female fertility index: 85.7-77.8-76.9-80.8
Gestation index: 100.0-100.0-100.0-100.0
Viability index on PND4: 99.1-95.6-99.2-94.4

Mating and Fertility Indexes were decreased in treated males and females compared to control males and females. Male fertility index of treated groups was reduced by 12.5% - 16.7.% - 12.5 % (100-300-1000 mg/kg/day) compared to control group; female fertility index of treated groups was reduced by 9.2% - 10.3% - 5.7% (100-300-1000 mg/kg/day) compared to control group. No dose response was recorded. Examination of sperms did not reveal changes in sperm motility, morphology and count, histopathological examination did not report pathological findings in genital tract of animals and spermatogenesis was not affected by the test item administration. The mating and fertility indexes in animal were lower but without conjunction with the related histopathological changes, sperm parameters and oestrous cycling, for this reason, this reduction of indexes alone cannot be considered toxicologically significant.
Gestation Index was the same in control and treated groups.
Viability Index (at PND4) was lower at the dose levels 100 (reduced by 3.5%) and 1000 (reduced by 4.7%) mg/kg/day in comparison with the control group. No dose dependency was recorded.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Cohort 1A and Cohort 1B
MALES and FEMALES: no treatment-related effects were detected during the health condition control and clinical observation in application period. The behaviour, clinical status and activity of treated animals were not affected by the test item administration.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Mortality of pups (totally per group) till the 4th day of lactation was 2-10-2-16 pups (0-100-300-1000 mg/kg/day). No mortality was observed after standardization (on day 4) till weaning (day 21).

FIRST GENERATION
Cohort 1A: there were no unscheduled deaths during the whole study.
Cohort 1B: two males (63/1B, 70/1B) were found dead on day 37 of application. This death was accidental (due to intubation error) not related to the test item administration
Cohort 1ex: there were no unscheduled deaths during the whole study

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistical evaluation of mean weight of the litters and pups was performed.
Statistically significantly changed body weight of male and female was recorded as follows:
- at the dose level 100 mg/kg/day decreased body weight of male and female pup was recorded on day 14 and 21;
- at the dose levels 300 mg/kg/day increased body weight of male pup on day 4 and decreased body weight of male and female pup on day 14.
-1000 mg/kg/day: No significant changes in body weight of male and female pup was recorded

Cohort 1A – Males
The statistic evaluation of body weight in all weeks of application and necropsy body weight did not show significant changes in treated males compared to control males.
The mean body weight of treated group of animals was slightly lower (statistically insignificantly) in comparison with the control group of males. The mean body weight increments of treated males of all dose levels were comparable with control for the whole time of application period.

Cohort 1A – Females
The statistic evaluation of body weight showed significant difference during the 3rd week of application only in females at the dose level 100 mg/kg/day in comparison with the control females. The mean body weight of animals of the dose levels 100 and 300 mg/kg/day was slightly lower (statistically insignificantly except 3rd week) in comparison with the control group for the whole application period. The mean body weight increments of treated females of all dose levels were similar with control for the whole time of application period.
Cohort 1B – Males
The statistic evaluation of body weight in all weeks of application and necropsy body weight did not show significant changes in treated males compared to control males.

Cohort 1B – Females
The statistic evaluation of body weight in all weeks of application and necropsy body weight did not show significant changes in treated females compared to control females. The body weight of treated females at all groups was similar to the control group of females

Surplus pups – Males
No significant changes were recorded. Statistically significantly decreased absolute weight of brain was recorded in males at the dose level 100 mg/kg/day. This change was accidental, not related to the test item administration. No significant changes of relative weights of organs treated males in comparison with control males were recorded.

Surplus pups – Females
No significant changes were recorded. No significant changes of absolute and/or relative weights of organs treated females in comparison with control females were recorded.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A and Cohort 1B:
MALES and FEMALES: the mean food consumption of treated males was comparable with the control group of animals.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A
MALES: fasted blood samples from a defined site were taken from ten males per dose group at termination. The selected haematological parameters were examined.
Statistically significantly changed parameters were recorded sporadically. The value of RBC was significantly increased in males at the dose level 100 and 1000 mg/kg/day. The significantly increased value of Hct in males at the dose level 100 mg/kg/day was associated with the increased value of RBC.
All changes were without toxicological importance and not related to the test item administration.

FEMALES: fasted blood samples from a defined site were taken from ten females per dose group at termination. The selected haematological parameters were examined.
Following statistically significantly changed values were recorded: increased value of Hct and percentual portion of eosinophiles (EOS) in females at the dose level 1000 mg/kg/day. Increased value of EOS (%) was recorded also in females at the dose level 100 mg/kg/day.
These changes are considered of no toxicological importance and did not relates to the test item administration.

Cohort 1A- Thyroid hormones
Blood (serum) samples from 10 males and 10 females of cohort 1A were assessed for thyroid hormones (T4 total and TSH).
No statistically significant differences in T4 and TSH concentration were noted between the treatment groups of males and females and the control groups.

Surplus pups
Blood (serum) samples from 10 animals/sex/group were assessed for thyroid hormones (T4 total and TSH). No statistically significant differences in T4 and TSH concentration were noted between the treatment groups and the control group.

Cohort 1B: not examinated

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A
MALES: fasted blood samples from a defined site were taken from 10 males per dose at termination. The selected biochemical parameters were examined.
Only one statistically significant change in measured parameters was recorded. The value of ALT was statistically significantly decreased in males at the dose level 1000 mg/kg/day in comparison to the control males. This change is considered as incidental and not related to the test item administration.

FEMALES: fasted blood samples from a defined site were taken from 10 females per dose at termination. The selected biochemical parameters were examined.
Statistically significantly changed value were not recorded.

Cohort 1B: not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A
MALES: statistically significantly decreased volume of urine was recorded in males at the dose level 100 and 1000 mg/kg/day. The presence of proteins and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.

FEMALES: statistically significantly decreased volume of urine was recorded in females at the dose level 1000 mg/kg/day The presence of leucocytes was recorded sporadically in females at the dose level 100 and 1000 mg/kg/day

Cohort 1B: not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

At all dose levels no differences in postnatal development of pups until the weaning (ear and eye opening, hair growth, tooth eruption, sucking ability) were observed in comparison with control group (see chapter 4.1.4.4 Postnatal development of locomotion and sensory development).
Also evaluation of data on sexual maturation (age and body weight at vaginal opening in female pups and balano-preputial separation in male pups) did not reveal significant changes in treated groups against control.

Sexual maturity

1) Preputial separation (PPS) in male pups and vaginal opening (VO) in female pups were observed ideally in 3 pups/sex/litter, i.e. in all pups from the cohort 1A, 1B and extra cohort 1ex.
Totally 471 animals (236 males and 235 females) were examined.
The preputial separation (PPS) in male pups was evaluated from PND30. PPS was determined by attempting to manually retract the prepuce using gentle pressure. The appearance of partial PPS was recorded in age 39.33/39.38/39.62/39.55 days (mean). The body weight on preputial separation day was 195.90/186.25/196.66/194.92 g in male pups (mean).
The vaginal opening (VO) in female pups was evaluated from the PND 30 and detected in age of 32.27/31.16/31.52/31.60 days (mean). The body weight on vaginal opening day was 117.70/105.03/109.37/111.27 g in female pups.

2) First estrus: Vaginal smears were examined daily for females Cohort 1A after the onset of vaginal patency, until the first cornified smear was recorded.
In all females cornified cells corresponding to an estrus were recorded within the few days after vaginal opening. Vaginal opening and first estrus in rats are almost simultaneous, as was the case in this study. The first estrus in females was recorded on 33.45/31.90/32.65/32.90 postnatal day (mean).

3) Oestrous cyclicity
Oestrous cycles was monitored for a period of two weeks (started on PND 75) in all F1-1A females. The cycle in rats lasts an average of four or five days.
No adverse effect of the test item on oestrous cyclicity was recorded The mean duration of cycle was 4.24/4.23/4.42/4.14 days. Females with irregular cycle (less than two cycle per two weeks and/or without cyclicity) were not included to the mean calculation. Number of females with irregular cycle was as follows: 2/1/1/0 (0/100/300/1000 mg/kg/day).

4) Observation of Sperm
Motility of sperms was examined in all 20 males per group. A comparison of sperm motility in the control males and males from treated groups did not show differences. The sperm morphology was evaluated in control males and males of the highest dose level at first instance. Because the treatment related changes were not recorded, the sperm morphology at doses 100 and 300 mg/kg/day were not evaluated. The test item treatment did not affect the sperm morphology.
The sperm count and viability of sperm was evaluated in 10 males per dose. Sperm count and viability were unaffected by the test item administration in treated males.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Corrected anogenital distance (anogenital distance relative to the cube root of body weight) did not show dose dependent response. Significant reducing of corrected AGD in treated males was not recorded. The values 2.04-2.06-2.03-2.01 in males and 1.29-1.18-1.20-1.27 in females, showed statistically significantly changed values only in females at the dose levels 100 and 300 mg/kg/day. The dose dependence was not recorded. The corrected distance of dosed females was decreased in dosed females at the dose levels 100 and 300 mg/kg/day compared to control animals, but it is not considered as a negative effect of the test item. All values are in a historical control range.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The presence of nipples in male pups was checked on day 13 of lactation and none were present (nipple presence in male pups on day 13 of lactation is undesirable).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Cohort 1A - Males: the statistical analysis did not reveal statistically significant differences in absolute weight of organs in the treated group of males in comparison to the control males. Sporadic changes in relative weight of organs – increased (statistically significantly) relative weight of epididymis and pituitary gland in males at the dose level 100 and 300 mg/kg/day were recorded. These changes did not related to the test item administration and were accidental.
Cohort 1A - Females: the statistical analysis of the absolute weight of organs revealed increased weight of brain (accidental finding). Evaluation of relative weight of organs revealed increased relative weight of ovaries in all dosed groups of females. This finding was statistically significant, but not dose-dependent, and not related to the test item administration.
Cohort 1B – Males: Statistically significant differences in absolute weight of organs were not recorded. Statistically significantly increased relative weight of epididymides was reported in males at the dose levels 100 and 300 mg/kg/day. This finding is not considered to be an adverse effect of test item administration.
Cohort 1B – Females: Statistically significant differences in absolute and relative weight of organs were not recorded.

SURPLUS PUPS (pups not selected for cohorts) Absolute and relative weight of brain, spleen and thymus in 10 randomly selected pup/sex/litter was determined.
Males: The statistical evaluation of necropsy body weight of males was performed. No significant changes were recorded. Statistically significantly decreased absolute weight of brain was recorded in males at the dose level 100 mg/kg/day. This change was accidental, not related to the test item administration. No significant changes of relative weights of organs treated males in comparison with control males were recorded.
Females: The statistical evaluation of necropsy body weight of females was performed. No significant changes were recorded. No significant changes of absolute and/or relative weights of organs treated females in comparison with control females were recorded

Gross pathological findings:

no effects observed

Description (incidence and severity):

Cohort 1A - Males
Control group: macroscopic findings were recorded only in one male. Congested stomach mucosa was reported. No macroscopic findings were recorded in other control male.
100 mg/kg/day: no macroscopic findings were recorded.
300 mg/kg/day: no macroscopic findings were recorded.
1000 mg/kg/day: decreased testes were recorded in one male. No macroscopic findings were recorded in other high dosed males

Cohort 1A - Females
Control group: no macroscopic findings were recorded.
100 mg/kg/day: no macroscopic findings were recorded.
300 mg/kg/day: no macroscopic findings were recorded.
1000 mg/kg/day: cyst on ovary was reported in one female. No macroscopic findings were recorded in other high dosed females.

Cohort 1B – Males and females
Control group: no macroscopic findings were recorded.
100 mg/kg/day: no macroscopic findings were recorded.
300 mg/kg/day: no macroscopic findings were recorded.
1000 mg/kg/day: no macroscopic findings were recorded.

Surplus pups: The macroscopic examination was performed in all pups not allocated to cohorts at necropsy day (PND22).
Control group: no pathological findings were recorded.
100 mg/kg/day: no pathological findings were recorded
300 mg/kg/day: no pathological findings were recorded.
1000 mg/kg/day: no pathological findings were recorded.

Cohort 1ex: no pathological findings were recorded.

Histopathological findings:

no effects observed

Description (incidence and severity):

Cohort 1A – No treatment-related changes were observed in male and female:
- Genital tract
- Liver
- Kidneys
- Pituitary gland
- Hemopoietic organs
In females the number of primordial and small growing follicles and corpora lutea were detected in F1 animals (10 females per group). Follicular enumeration was conducted on control and high-dose females at the first step. Because no treatment related effect was detected, lower doses were not examined. The mean number of follicles of high dosed females was slightly higher in comparison with the control females. Statistically significant differences were not recorded. The mean number of corpora lutea was lower (about 13.6%) in a group of high dosed females in comparison with the control females. This decrease was statistically insignificant. (According to OECD 151: “Where high dose group counts are not less than 85% of control and are not statistically significantly different, no further evaluation is considered necessary”)

Cohort 1B: Histopathological examination was not performed (the result of cohort 1A were not unclear). Following tissues were processed to the block stage: testes, epididymides, prostate, seminal vesicles, vagina, uterus, ovaries and pituitary gland.

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

First, the development of the pups as a whole litter was examined on day 1 postpartum and subsequently the postnatal development of locomotion and sensory development of pups were examined on days 4, 7, 14 and 21.

On day 1 body temperature of whole litter (indicative), the ability to suck, issuing of sound and ability of pup to turn from back to the abdomen were examined.
No significant differences in mean body temperature (36.42/36.90/36.78/36.63°C) among the control and dosed groups of animals were recorded. Ability of sucking of pups and ability to issue a sound was recorded in all pups. Ability of pup to turn from back to abdomen was observed in all pups from control and dosed groups.
Only one exception was recorded. All pups from litter of the female No.311 were born live, but were cold, they did not suck milk and issued sound. Fourteen of fifteen pups died till the second day postpartum, and cannibalism of the last pup was recorded till the third day postpartum. This case is not related to the test item administration, it was a consequence of difficult parturition (see chapter. 4.1.2.4 Health condition control and clinical observation on page 42 and chapter 4.1.2.5 Detailed Clinical Observation on page 43 of this report).
On day 4, tilting of ears and “pivoting” (forelimbs act as paddles) were examined in pups.On day 7, growth of hair and the crawling were examined in pups.
On day 14, opening of eyes, teeth pruning (upper incisors) and coordinate walking were examined.
On day 21, pruning molars, growth of hair (also on the genitals) and ability of running were examined.
No differences in development of pups among the control group and dosed groups were recorded.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 1A
For the investigation of pre- and postnatally induced immunotoxic effects, 10 male and 10 female animals from each group were examined.
The weighing of the lymph nodes associated with route of exposure (paraaortal lymph nodes) and distant from the route of exposure (axillary lymph nodes) was performed.
Both in MALES and FEMALES no statistically significant changes in absolute and relative weight of axillary and paraaortal lymph nodes were detected in treated animals compared to control animals
Percentage representation of T-; B-lymphocytes and NK cells in spleen of treated animals were comparable with the control animals. Statistically significant differences were not detected except a significantly decreased ratio of CD8+ T lymphocytes in males at all dose levels. Dose dependency was not recorded. Due to the absence of dose dependency, histological findings and altered spleen weight, this finding was considered to be not toxicologically significant. The ratio CD4+/CD8+ in treated groups of males and females was not significantly changed in treated animals against the control animals.
All monitored parameters related to the evaluation of the immunotoxicity of the test item (lymph node weight, spleen weight, evaluation of splenic lymphocyte populations and histopathological evaluation) did not show an immunotoxic effect of the test item on dosed animals.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

histopathology: neoplastic

developmental neurotoxicity

developmental immunotoxicity

other:

Remarks on result:

other: The test is not complete and some examinations are on going.

2

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

histopathology: neoplastic

developmental neurotoxicity

developmental immunotoxicity

other:

Remarks on result:

other: The test is not complete and some examinations are on going.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Any other information on results incl. tables

The statistical evaluation of the number of live pups (on the first check of litter, the 4^th and 21^st day after parturition) was performed. No statistically significant changes were recorded. The total number of live pups on the first check of litter and on the 4^th day before standarditazion at all dose levels was lower in comparison with the control group. The lowest number of pups was recorded in group of females at the dose level 100 mg/kg/day. The mean number pups per litter at all dose levels was also lower compared to control. The total number of live pups and mean number of live pups per litter after standardisation on day 7, 14, 21 after parturition at all doses were slightly lower in comparison with the control group Total number of stillborn and/or dead pups was the highest in control group.

See attached tables for results. 

 

Applicant's summary and conclusion

Conclusions:

NOAEL for SYSTEMIC TOXICITY = 1000 mg/kg/day for P and F1 males and female
NOAEL for REPRODUCTION and DEVELOPMENT = 1000 mg/kg/day

Executive summary:

The test item, sodium (xylenes and 4-ethylbenzene) sulfonate , was assessed to evaluate specific life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal exposure to test item. The test was performed follwing OECD 443, Extended One Generation Reproductive Toxicity Study. 

Systemic toxicity
Ten weeks premating exposure duration for the parental (P) generation was ordered. The length of the premating exposure period was ten weeks to cover the full spermatogenesis and folliculogenesis before the mating. Mortality, body weight, food consumption, health condition, clinical status after application in treated parental males and females and animals of F1 generation were unaffected by treatment of the test item.

During the haematological, biochemical analysis and urinalysis, no significant treatment related changes were recorded. No adverse treatment related effects of the test item were recorded during the biometry of organs and histopathological examination.  

Sperm analysis did not reveal changes in sperm motility, morphology and count in groups of treated P-males compared to control P-males. Sperm viability in treated groups of P-males (% viability of sperm is calculated based on the number of live cells relative to the total number of cells in the of sperm suspension from caudal epididymis) revealed decrease at the dose level 300 and 1000 mg/kg/day. In spite of this, the male rats’ ability to produce sperm that can fertilise eggs was maintained. The examination of sperm viability in F1 males (treated for 10 weeks) in all treated groups reported the same values of sperm viability in treated groups as in the control group (actually the viability in treated males was better than in control males). The sperm analysis of F1 males did not reveal changes in sperm motility, morphology, count and viability between the treated and control group of males. Because the sperm viability of all treated groups of F1-males (after 10 weeks application) was the same and even better then viability of control group, histological examination did not reveal pathological finding and the spermatogenesis was not affected by the test item administration, the decreasing of sperm viability in P-males was not considered adverse effect of the test item.

Reproduction toxicity
Mating and fertility indexes of P generation of animals were lower in treated groups of animals as follows: male and female mating indexes were 100.0%-96.4%-92.9%-92.9% (dose levels 0-100-300-1000 mg/kg/day); male fertility indexes were 85.7%-75.0%-71.4%-75.0% and female fertility indexes were 85.7%-77.8%-76.9%-80.8%. Mating indexes of treated groups were reduced by a maximum of 7% in comparison with the control group. Male fertility index of treated groups was reduced by 12.5% - 16.7.% - 12.5 % (100-300-1000 mg/kg/day) compared to control group; female fertility index of treated groups was reduced by 9.2% - 10.3% - 5.7% (100-300-1000 mg/kg/day) compared to control group. No dose response was recorded. The mating and fertility indexes in animal were lower but without conjunction with the related histopathological changes, sperm parameters and oestrous cycling, for this reason, this reduction of indexes alone cannot be considered toxicologically significant.
All pregnant females gave birth to pups. No abortion was recorded. The number of stillborn pups was the highest at the control group (15-7-6-4 respectively)
Post-implantation losses were the highest at control group (2.75-1.57-2.10-2.43).
A lower mean number of implantations was recorded in treated groups 100 and 300 mg/kg/day of females, 17.25-13.52-14.60-16.86 respectively (statistically significant decrease at the dose levels 100 and 300 mg/kg/day); on the other hand, at the dose level 1000 mg/kg the mean number of implantation was similar to control. Decreased number of implantation in low and medium dose level is associated with lower mean number of live pups born in treated groups (14.50-11.95-12.50-14.43). Decreased number of pups was reported mainly at the dose level 100 and 300 mg/kg/day but statistical evaluation did not show significance. A mean number of pups in treated groups was reduced by 17.6% - 13.8% - 0.5% (100-300-1000 mg/kg/day). The mean number of pups at the dose level 1000 mg/kg was similar to the control. No dose response was recorded.

Because dose response (which provide more confidence and is more indicative of a chemically mediated effect) was not reported, sperm parameters (motility, morphology and count) and histological evaluation of reproductive organs did not reveal  pathological effect of the test item, a reduced number of implantation in low and medium dose level is not considered as adverse effect of test item administration.

The administration of the test item did not affect reproductive organs. Spermatogenesis in the testes of the high dose administered P and F1 males was without any pathological findings. No treatment-related changes were found in the male genital tract of both generations of males. No treatment-related histological changes were found in the female genital tract of both generations. Ovarian follicle count was higher in females at the dose level 1000 mg/kg/day than in females of control group. Also corpora lutea count  in the high dosed F1 females was not adversely affected by the test item administration.

The number of pregnant females, mating and fertility indexes were lower in treated groups but without conjunction with the related histopathological changes, sperm parameters and oestrous cycling. For this reason, these reduction of indexes alone cannot be considered toxicologically significant.

Development
Postnatal development of pups was not affected by the test item treatment. The observed parameters of the pups of treated mothers were similar to the control pups. The sexual maturity of F1 animals were unaffected by the test item administration.

Immunotoxicity of the test item was not recorded in either the parental animals or in animals of F1 generation.