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Toxicological information

Endpoint summary

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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The ASA Category comprises the following 5 aromatic sulphonic acids:


TSA, Toluene-4-sulphonic acid (EC 203-180-0, CAS 104-15-4)
XSA, (Xylenes and 4-ethylbenzene) sulphonic acid (EC 701-247-3, CAS -) Former EC 246-839-8
CSA, p-cumene sulphonic acid (EC 240-210-1, CAS 16066-35-6)
BSA, Benzene sulphonic acid (EC 202-638-7, CAS 98-11-3)
HBSA, 4-hydroxybenzensulphonic acid (EC No. 202-691-6, CAS No. 98-67-9)


The toxicity to reproduction and development was assessed with available test on ASA and related salt (Hydrotropes category). Hydrotropes are considered similar to ASA and could be use as read across.


BSA was tested under OECD 422 extended to 90 days toxicity and the NOAEL for reproduction and developmental toxicity was established as 500 mg of active ingredient /kg body weight/day which is the highest tested dose. This highest dose was selected based on the effects on the repeated dose part of the study. All changes in reproductive parameters observed in parental males and females at all dose levels were considered to be of no toxicological significance.


There are available studies for the chemically related hydrotrope substances that looked at reproductive organs. Hydrotropes are the salt form of the sulphonic acids and therefore are used as a support for this endpoint.


The study on STS performed under OECD 421 did not show any effects. The 90-day oral rat and oral mouse studies and the 2-year chronic dermal rat and mouse studies on SXS included examination of sex organs of both sexes. No treatment related effects on reproductive organs were reported at doses. A new OECD 443 was performed on SXS which is the most representative member of the hydrotropes category since it contains a significant proportion of both mono-alkyl (ethyl) and di-alkyl (dimethyl) chemical species and thus represents both the mono-alkyl and di-alkyl substances contained within the category.


The available results on reproductive toxicity are:  
STS, OECD 421 (2004): NOAEL = 1000 mg a.i./kg bw              
BSA, OECD 422 extended to 90 days (2020): NOAEL= 500 mg a.i./kg bw                     
SXS, OECD 443 (2021): NOAEL = XX mg a.i./kg/bw


The NOAEL value used for the risk assessment for the reproduction toxicity is 500 mg/kg bw from the OECD 422 extended to 90 days toxicity performed on BSA.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
extended to 90 days toxicity; microclimatic deviation of humidity which did not affect teh results of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal species: laboratory rat
Strain: Wistar CRL (SPF quality - guaranteed)
Supplier: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
Sex: males and females
Age of animals: males, females: sexually adult, 8 weeks on arrival, Dose-range finding experiment: 8 weeks on arrival
Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species and because rat is recommended according to the test guideline
Acclimatization: Main study: 12 days
Number of animals main study: 12 females and 12 males per group, 6 males and 6 females per satellite group
Housing conditions:
Main study: SPF conditions according to internal SOP No.12
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, satellite animals - 2 rats of the same sex in one cage
Food: maintenance pelleted diet for rats and mice - Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law
Light cycle: 12 hour light / 12 hour dark
Microclimate: 22 ± 3 °C, relative humidity 30-70 %
Bedding: sterilized soft wood fibers Lignocel
Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight
Identification of animals: the animals have been 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
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The item was supplied as a 70.9 % w/w Total Active Acid aqueous solution and the dose levels of the test item was recalculated on 100 % of active ingredient BSA.
Details on mating procedure:
Animals were mated from the 58th day of study. Mating 1:1 (one male to one female) was used in this study. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item application forms were determined by measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility. Measuring was performed on two concentrations of application form: 10 mg of a.i./10 mL and 1000 mg of a.i./10 mL.

Homogeneity
The samples were taken after 30 minutes of mixing by magnetic stirrer (500 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content for the determination of homogeneity of both application forms. Two samples were taken from each place.

Stability
The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals. Time interval 0 min represents for both concentration levels the time after 20 minutes of mixing by magnetic stirrer at 500 rpm.

The results of analyses (homogeneity and stability) showed that both application forms of the test item, benzenesulphonic acid (BSA), (10 mg and 1000 mg /10 mL), at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Based on active ingredient
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Based on active ingredient
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Based on active ingredient
No. of animals per sex per dose:
12 females and 12 males per group; 6 males and 6 females per satellite group.
Control animals:
yes, concurrent vehicle
Details on study design:
A dose-range finding experiment (DRFE) was performed to determine the dose levels for the main study. The following examinations were performed in DRFE: body weight, clinical examinations, haematological examination and pathological examination of animals. The results were collected in DRFE Summary Report which constitutes the annex to the study final report (Annex 2). Results were used for establishing the doses for main study.

In the main study all animals are administered the test item before mating, during mating, during the pregnancy and lactation period. Parental males and satellite animals (males and females) have a total of 90 days of administration; the parental females are administered until the end of the lactation period (until the 13th post-natal day of pups). Parental females have a minimum of 90 days of administration, but usually it is a few days longer - depending on which day they become pregnant. In this study the maximum for females was 99-93-103-106 days of administration at the dose levels 0-125-250-500 mg of a.i./kg bw/day respectively.

The first six males, six mothers with delivered pups per group and satellite groups of animals (control and treated) are a part of the Repeated dose toxicity part of study and examined with the respect to toxicity effect of the test item on organism (functional observations, ophthalmological examination, haematology, biochemistry, full biometry and full pathology were performed in 6 males + 6 females of each dose level and in satellite animals). Satellite animals are used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effect, for 28 days post treatment.
All twelve males and females per group are a part of the Reproduction part of study and examined with the respect to reproduction parameters (biometry of reproduction organs, sperms analysis, and examination of uteri, pups and litters parameters).
At the end of the main study, NOAELs were determined for Repeated dose Toxicity, Reproduction and for Development.
Parental animals: Observations and examinations:
Health condition control: daily - during the acclimatisation and the experimental part
Body weight: males and satellite animals - the first day of administration and then weekly and before necropsy
females - the first day of administration and then weekly,
during pregnancy: 0, 7th, 14th, 20th day,
during lactation: 1st, 4th day, 12th day and 13th day
pups (litters) - 1st, 4th day, 12th day and 13th day
pups – individually – 4th day of lactation
Food consumption: weekly and on the same days as body weight (except the mating period)
satellite males and females – weekly
Water consumption: satellite males and females – three times a week
Clinical observations: males and females - daily during the administration period
pups - as soon as possible after delivery and then daily
Mortality control: twice daily
Detailed clinical observation: efore the first application and then weekly (except the mating period)
Functional observation: at the end of administration/observation period = last week of the study

Laboratory examinations:
- urinalysis: the last day of administration/observation period – only males
- haematology: males – after the end of application period
parental females – on the 13th day of lactation
satellite animals – after the end of observation period
- biochemistry: males and nonpregnant females – after the end of application period
2 pups per litter - on the 4th day of lactation
parental females and pups – on the 13th day of lactation
satellite animals – after the end of observation period
- anogenital distance measurement: pups – 4th day of lactation

Oestrous cyclicity (parental animals):
Vaginal smears were monitored at the 4th and 5th weeks of the application period for monitoring of oestrous cycle of females. No females with irregular oestrus cycle were detected.
Sperm parameters (parental animals):
In all males of the main groups, the sperm parameters, sperm motility and sperm morphology were examined.
Litter observations:
All pups were observed in natural conditions in their cages daily during the lactation. Changes inAny behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and then daily till 13th day ofd lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Postmortem examinations (parental animals):
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded (Repeated Dose Toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (Reproduction part of study – all animals).
Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight. From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.
Postmortem examinations (offspring):
Pathological examination and thyroid gland weight.
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used.
Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group. The results statistically significant on probability level (p ≤ 0.05) were indicated in the summary tables.

In general:
The parametric tests were used for statistical evaluation of
• body weight of males and females
• mean pup weight
• litter weight
• anogenital distance of pups
• selected haematology parameters
• blood biochemistry parameters
• data from urinalysis (pH, volume)
• data from biometry of organs
As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed then the transformation of data was performed (Box-Cox transformation). If still the normal distribution was not achieved, non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.
For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).

The non-parametric tests were used for statistical evaluation of
• selected reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of implantations)
• selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)
The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤ 0.05).
Reproductive indices:
Post-implantation loss, post-natal loss, Male and female mating index, male and female fertility index and Gestation index
Offspring viability indices:
Viability index on PND4
Clinical signs:
no effects observed
Description (incidence and severity):
No changes of animal health status and clinical symptoms of intoxication were observed in all treated animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male at the dose level 500 mg of a.i./kg bw/day died on the 58th day of application due to gavage error. This death is not related to the test item application.
One female at the dose level 250 mg of a.i./kg bw/day died on the 52nd day of application due to gavage error.
One female at the control group died on the 83rd day of application due to complication during delivery.
These deaths are not related to the test item application.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Decreased body weight in males at the highest dose level was recorded during the application period. In females at the highest dose level increased body weight at the 20th day of pregnancy was recorded
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effect of the test item on the food consumption was recorded in both sexes during the study.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
An incidence of cannibalism of pups was recorded. This finding was incidental and not caused by the test item administration.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No findings related with the test item treatment were detected during the histopathological examination of reproductive organs and pituitary gland in animals at the highest dose level.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Hormones T4, T3 and TSH values were similar between control and tested group.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No significant changes in sperm motility and morphology.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male and female mating index was decreased at the mid dose level. Male and female fertility index was increased at the mid and highest dose levels. The gestation index was increased in females at the lowest, mid and highest dose levels in comparison with the control. The post-implantation and post-natal losses were not significantly changed in treated and control groups.
Number of females showing evidence of copulation was decreased at the mid dose level. In two females from this group, presence of sperm was not detected during the mating period. Mean duration of mating (days) was increased at the mid dose level.
Number of treated females achieving pregnancy was similar in comparison with the control females.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of live pups at the first litter check after parturition, on the 4th day and 13th day of lactation was increased at all dose levels compared to the control group.
One stillborn pup in one control female was recorded. Mean numbers of pups per litter at the dose level 500 mg of a.i./kg bw/day was increased at all examination intervals.
Body weight and weight changes:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Blood samples from the 13-day old pups were assessed for serum levels of thyroid hormones (T4, T3 and TSH). Pup blood was pooled by litter.
No statistically significant differences were recorded in the mean concentration of hormone T4, T3 and TSH in pups from treated groups against control pups
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease of mean of anogenital distance in male pups at the middlemid and highest dose levels was recorded (without dose dependence). In male pups at the highest dose level statistically significantly decreased corrected anogenital distance was recorded. Examination of concentration of T4, T3 and TSH hormones and histopathological examination of thyroid gland no showed significantly changes, therefore decreased of the corrected anogenital distance was not considered to be of biological or toxicological significance.
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weight of the thyroid gland was not statistically significantly changed in male and female pups of treated mothers in comparison with the control group of mothers.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):
No macroscopical findings were recorded in other pups/litters.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
Critical effects observed:
no
Reproductive effects observed:
no

Table No. 38: Body weight and body weight increment of males – Reproduction toxicity

Meanbody weight and body weight increment

MALES

Group mean body weight±standard deviation

(grams)

Mean body weight increment(grams/animal/day)

Application

period

Group code

Application period

Group code

0

125

250

500

0

125

250

500

Before start of administration

398.98

±

28.50

399.77

±

17.92

401.33

±

11.85

398.83

±

15.34

Before start of administration

-

-

-

-

1stday of administration

415.99

±

33.39

405.59

±

19.47

408.63

±

12.48

401.78

±

14.50

1stday of administration

-

-

-

-

1stweek

440.96

±

38.39

426.63

 ±

23.04

433.67

±

15.69

422.32

 ±

18.37

1stweek

3.57

3.01

3.58

2.93

2ndweek

461.98

 ±

39.62

445.02

±

25.37

451.56

±

19.30

441.71

±

18.94

2ndweek

3.00

2.63

2.56

2.77

3rdweek

480.03

±

44.96

465.78

±

29.07

473.82

±

22.60

455.18

±

20.21

3rdweek

2.58

2.97

3.18

1.92

4thweek

502.31

±

50.68

489.54

±

31.06

490.78

±

22.90

475.28

±

23.77

4thweek

3.18

3.39

2.42

2.87

5thweek

525.23

 ±

52.70

509.08

±

34.41

511.43

±

27.11

497.46

±

25.31

5thweek

3.27

2.79

2.95

3.17

6thweek

535.19

±

58.56

522.58

±

38.00

526.72

±

30.81

509.26

±

26.07

6thweek

1.42

1.93

2.18

1.68

7thweek

549.80

 ±

60.00

537.93

±

38.11

539.72

±

31.21

520.40

±

28.31

7thweek

2.09

2.19

1.86

1.59

8thweek

564.63

±

68.66

549.48

±

42.54

546.52

±

32.21

529.75

±

34.68

8thweek

2.12

1.65

0.97

1.34

9thweek

562.87

±

63.64

553.13

±

39.46

550.03

±

35.38

534.49

±

36.38

9thweek

-0.25

0.52

0.50

0.68

10thweek

578.93

±

65.11

563.01

±

41.99

563.16

±

33.60

545.36

±

38.13

10thweek

2.29

1.41

1.88

1.55

11thweek

588.93

±

72.55

568.45

±

44.79

568.08

±

35.25

550.37

±

41.05

11thweek

1.43

0.78

0.70

0.72

12thweek

602.60

±

73.97

573.88

±

46.72

577.06

±

34.65

558.30

±

40.34

12thweek

1.95

0.78

1.28

1.13

13thweek

603.97

±

76.77

586.47

±

51.91

585.41

±

35.09

571.65

±

45.38

13thweek

0.27

2.52

1.67

2.67

Total body weight gain (g)

(week 13 – 1stday of administration)

187.98

180.88

176.78

169.87

 

Necropsy body weight*

563.75

 ±

72.02

548.98

±

45.58

544.88

±

31.18

524.22

±

42.66

Note:Different body weights on the 1stday of administration was caused by sequential inclusion of 

         animal groups to the study.

         * The loss of body weight of all males was caused by fastingbefore blood collection

Table No. 39: Food consumption of males – Reproduction toxicity

Mean food consumption per dose group - MALES

(grams/animal/day)

Group code

0

125

250

500

29.04

28.44

28.64

29.11

28.63

28.18

28.06

29.11

27.78

27.79

27.70

28.21

28.02

28.03

27.62

28.54

29.54

29.20

28.73

29.49

28.78

28.41

28.21

28.79

28.89

28.72

28.10

28.55

29.35

28.11

27.45

28.30

Mating period

29.60

27.92

28.35

29.05

30.09

27.99

28.08

29.35

29.41

28.84

27.85

28.60

Table No. 40: Weight of organs of males – Reproduction toxicity

Mean weight of organs - MALES

 Organs

Group code

0

125

250

500

Absolute weight

 (g±SD)

Relative weight

(%±SD)

Absolute weight

(g±SD)

Relative weight

(%±SD)

Absolute weight

(g±SD)

Relative weight

(%±SD)

Absolute weight

(g±SD)

Relative weight

(%±SD)

EPIDIDYMIS

0.8992

±

0.1008

0.1668

±

0.0261

0.9072

±

0.0763

0.1656±

0.0110

0.9544

±

0.0730

0.1753

±

0.0115

0.8942

±

0.0746

0.1714

±

0.0180

TESTES

4.6979

±

1.0119

0.8669

±

0.1900

4.3295

±

0.2840

0.7924±

0.0706

4.4620

±

0.2316

0.8202

±

0.0445

4.4152

±

0.6246

0.8456

±

0.1220

 PROSTATE GLAND + SEMINAL VESICLES

4.7194

±

0.3514

0.8798

±

0.1473

4.6743

±

0.3767

0.8563

±

0.0902

4.2311

±

0.3707

0.7780

±

0.0704

4.1966

±

0.3885

0.8077

±

0.1199

 PITUITARY GLAND

0.0162

±

0.0018

0.0030

±

0.0005

0.0155

±

0.0015

0.0028

±

0.0004

0.0155

±

0.0015

0.0028

±

0.0003

0.0152

±

0.0016

0.0029

±

0.0004

THYROID GLAND

0.0322

±

0.0012

0.0060

±

0.0008

0.0321

±

0.0009

0.0059

±

0.0005

0.0321

±

0.0013

0.0059

±

0.0004

0.0324

±

0.0012

0.0062

±

0.0005

Note:SD – standard deviation

         grey field= values statistically significant (p ≤ 0.05)

Table No. 41: Observation of sperm

Observation of sperm

Observed parameters

Group code

0

125

250

500

Sperm motility

(mean grade)

1.08

1.08

1.00

1.00

Sperm motility

(grade/number of males)

1/11

2/1

1/11

2/1

1/12

1/12

Sperm morphology

(percentage of affected sperms - mean)

6.33

6.33

6.92

6.67

- Affected head

2.17

2.25

2.58

2.67

- Bent neck

1.75

1.58

1.58

1.42

- Bent tail

2.42

2.50

2.75

2.58

Note: Sperm motility - grade: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm; e.g. 1/8 = fast progressive motility in 8 males

Table No. 42: Concentration of T4, T3 and TSH hormones of males

Group code

0

125

250

500

6.459 ± 1.121

6.489 ± 1.933

7.673 ± 2.042

6.001 ± 0.866

0.501 ± 0.072

0.515 ± 0.124

0.581 ± 0.125

0.529 ± 0.060

1.171 ± 0.668

0.773 ± 0.664

0.661 ± 0.242

0.952 ± 0.817

Table No. 43: Histopathological findings of males – Reproduction toxicity

Organ/Diagnosis

Group code

0

125

250

500

Number of examined animals

12

0

0

11

Mortality

0

0

0

1

Without pathological finding

                                  of reproductive organs

4

/

/

6

Testes:atrophy of tubules

3

/

/

0

Testes:cellular detritus in sporadic tubules

1

/

/

0

Testes:dilatation of tubules

0

/

/

1

Testes:degeneration of germinal epithelium with production of large multicleated spermatid

0

/

/

1

Epididymides: lymphocytic infiltration of interstitium

1

/

/

2

Prostate gland:dilatation of acins

2

/

/

0

Prostate gland:focal atrophy of acins with coagulation and mineralization of their content

0

/

/

1

Prostate gland:focal atrophy of acins

1

/

/

0

Prostate gland:focal polymorphonuclear inflammation of acins

1

/

/

0

Prostate gland:focal lymphocytic infiltration of interstitium

0

/

/

1

Table No. 44: Body weight/body weight increment of females – Reproduction toxicity

Meanbody weight and body weight increment

FEMALES

       Mean body weight

                                       (grams)

Mean body weight  

     increment

(grams/animal/day)

Application period

Group code

Application period

         Group code

0

125

250

500

0

125

250

500

Before mating

Before start of administration

240.19

±

14.91

241.54

±

8.38

241.89

 ±

15.45

239.88

 ±

12.88

Before start of administration

-

-

-

-

1stday of administration

249.69

±

14.71

243.03

±

11.03

241.79

±

14.85

243.88

±

12.94

1stday of administration

-

-

-

-

1stweek

257.07

±

14.20

251.63

±

10.95

253.33

±

15.23

253.28

±

14.45

1stweek

1.05

1.23

1.65

1.34

2ndweek

265.54

±

14.99

265.89

±

13.53

261.57

±

16.83

264.35

±

11.83

2ndweek

1.21

2.04

1.18

1.58

3rdweek

272.98

±

16.21

271.02

±

12.29

267.18

±

17.76

267.48

±

12.11

3rdweek

1.06

0.73

0.80

0.45

4thweek

279.38

±

17.34

273.57

±

12.65

271.50

±

17.90

277.23

±

13.46

4thweek

0.91

0.36

0.62

1.39

5thweek

286.32

±

18.47

281.22

±

14.26

278.38

±

17.96

283.73

±

11.20

5thweek

0.99

1.09

0.98

0.93

6thweek

288.91

±

19.35

283.31

±

11.94

281.66

±

18.91

284.77

±

14.37

6thweek

0.37

0.30

0.47

0.15

7thweek

292.54

±

21.87

290.38

±

14.75

286.96

±

18.53

288.58

 ±

14.61

7thweek

0.52

1.01

0.76

0.54

8thweek

295.35

±

19.56

290.01

±

15.89

286.05

±

17.90

293.13

±

15.25

8thweek

0.40

-0.05

-0.13

0.65

Total body weight gain (g)

(week 8 - 1 day of administration)

45.66

46.98

44.26

49.25

 

9th– 10thweek

Mating period

9th– 10thweek

Mating period

Day of pregnancy

0

300.89

±

14.57

304.50

±

12.55

290.05

±

11.70

303.47

±

16.99

0

-

-

-

-

7

325.41

±

16.72

320.74

±

17.61

313.41

±

11.69

332.19

±

12.85

7

3.50

2.32

3.34

4.10

14

358.53

±

20.71

362.29

±

22.52

344.68

±

9.15

367.94

±

17.79

14

4.73

5.94

4.47

5.11

20

429.54

±

48.84

434.18±

36.27

410.81

±

50.97

453.99

±

27.34

20

11.84

12.54

12.52

14.34

Total body weight gain (g)

(20 - 0 day of pregnancy)

128.65

129.68

120.76

150.52

 

Day of lactation

1

342.83

±

23.89

349.45

±

13.45

328.65

±

12.72

359.23

±

17.84

1

-

-

-

-

4

356.39

±

27.42

363.55

±

20.55

348.35

±

21.25

381.94

±

12.90

4

4.52

4.70

6.57

7.13

12

385.74

±

39.32

392.26

±

19.23

371.81

±

38.15

395.83

±

12.55

12

3.67

3.59

2.93

1.74

13*

308.92

±

26.02

310.65

±

19.93

303.74

±

21.89

318.87

±

28.35

13*

-

-

-

-

Total body weight gain (g)

(12 - 1 day of lactation)

42.91

42.81

43.16

36.60

 

Note:* - the loss of body weight of all mothers was caused by fastingbefore blood collection

Table No. 45: Food consumption of females – Reproduction toxicity

Mean food consumption per dose group - FEMALES

(grams/animal/day)

Application period

Group Code

0

125

250

500

Before mating

1stweek

19.34

20.04

19.96

19.49

2ndweek

19.64

19.98

19.73

20.20

3rdweek

19.23

19.34

19.33

19.84

4thweek

18.57

19.03

19.78

20.15

5thweek

19.21

19.80

19.46

19.62

6thweek

17.96

18.85

19.71

19.39

7thweek

18.36

19.56

19.54

18.38

8thweek

18.42

18.72

18.91

19.87

 

9th– 10thweek

Mating period

Day of pregnancy

0 - 7

20.45

19.25

19.74

21.25

7 - 14

23.68

24.89

25.68

27.00

14 - 20

27.63

29.64

28.34

30.99

Day of lactation

1 – 4

40.93

34.50

32.74

44.74

4 - 12

55.49

56.61

58.74

61.57

Table No. 46: Weight of organs of females – Reproduction toxicity

Mean weight of organs - FEMALES

Group code

 

0

125

250

500

Organs

Absolute weight

 (g±SD)

Relative weight

(%±SD)

Absolute weight

(g±SD)

Relative weight

(%±SD)

Absolute weight

(g±SD)

Relative weight

(%±SD)

Absolute weight

(g±SD)

Relative weight

(%±SD)

 OVARIES

0.1537

±

0.0212

0.0492

±

0.0085

0.1540

±

0.0213

0.0481

±

0.0070

0.1354

±

0.0141

0.0395

±

0.0150

0.1357

±

0.0181

0.0413

±

0.0062

 UTERUS

0.7378

±

0.2125

0.2371

±

0.0790

0.6072

±

0.0818

0.1894

±

0.0247

0.6258

±

0.1448

0.1824

±

0.0830

0.5740

±

0.0978

0.1749

±

0.0336

 PITUITARY GLAND

0.0191

±

0.0009

0.0061

±

0.0008

0.0176

±

0.0017

0.0055

±

0.0006

0.0162

±

0.0016

0.0047

±

0.0018

0.0170

±

0.0014

0.0052

±

0.0007

THYROID GLAND

0.0308

±

0.0006

0.0098

±

0.0009

0.0308

±

0.0013

0.0096

±

0.0004

0.0311

±

0.0012

0.0090

±

0.0033

0.0314

±

0.0014

0.0095

±

0.0008

Note:SD – standard deviation grey field= values statistically significant (p ≤ 0.05)

Table No. 47: Histopathological findings of females – Reproduction toxicity

Organ/Diagnosis

0

125

250

500

Number of examined animals

11

0

0

12

Mortality

1

0

0

0

Without pathological finding

                                  of reproductive organs

8

/

/

9

Uterus – cervix:eosinophile cells infiltration of mucosa

2

/

/

0

Uterus:eosinophile infiltration and dilatation

1

/

/

0

Uterus:hyperplasia of endometrium

0

/

/

1

Uterus: hydrometra (non-pathological change)

1

/

/

1

Ovary:angiectasia of the rete ovarii

0

/

/

1

Pituitary gland:hemangiectasia

0

/

/

1

Table No. 48: Number of pups

Number of pups

Parameter
Group Code
0
125
250
500

Total number (all pups)

at first check of litter after parturition

72

97

92

131

Total number of live born pups

male/female

71

39M/32F

97

49M/48F

92

45M/47F

131

57M/74F

Total number of stillborn pups

1

0

0

0

Total number of live pups at 4thday of lactation

male/female

70

39M/31F

96

49M/47F

87

43M/44F

130

56M/74F

Total number of live pups at 13thday of lactation

male/female

62

35M/27F

82

42M/40F

75

37M/38F

111

47M/64F

Note:M - male pups, F – female pups

The difference in the number of pups between the 4thand 13thday of lactation was caused by the sacrificing of pups for blood sample collection (for eventual T4 analysis on 4thday of lactation) and due to the death and/or cannibalism of pups.

Table No. 49: Mean number of pups

Mean number of pups per litter

Parameter
Group Code
0
125
250
500

Mean number of live pups at first check

of litter after parturition

10.14

12.13

11.50

14.56

Mean number of live male/female pups

5.57/4.57

6.13/6.00

5.63/5.88

6.33/8.22

Mean number of stillborn pups

0.14

0.00

0.00

0.00

Mean number of live pups at 4thday of lactation

10.00

12.00

10.88

14.44

Mean number of live male/female pups

5.57/4.43

6.13/5.88

5.38/5.50

6.22/8.22

Mean number of live pups at 13thday of lactation

8.86

10.25

9.38

12.33

Mean number of live male/female pups

5.00/3.86

5.25/5.00

4.63/4.75

5.22/7.11

Table No. 50: Mean body weight of pups/litter

Mean body weight

 (grams)

 

Group code

Day of study

0

125

250

500

Litter

Pup

Litter

Pup

Litter

Pup

Litter

Pup

1stday

first check

of litter after parturition

70.74

6.58

83.60

7.05

78.01

6.62

97.98

6.77

4thday of lactation

126.98

10.93

126.01

10.42

129.34

10.22

151.86

10.61

12thday of lactation

298.10

30.41

274.56

28.06

293.51

26.53

336.73

27.75

13thday of lactation

310.50

31.46

284.24

28.90

299.71

27.63

342.72

28.39

Table No. 51: Anogenital distance

Day 4 - Mean anogenital distance

(mm)

Group code

0

125

250

500

Male

Female

Male

Female

Male

Female

Male

Female

4.06

2.69

3.88

2.48

3.74

2.47

3.80

2.58

11.09

10.63

10.62

10.19

10.55

10.35

11.02

10.36

1.82

1.23

1.77

1.14

1.73

1.15

1.71

1.18

Note: grey field= values statistically significant (p ≤ 0.05)

Table No. 52: T4, T3 and TSH hormones concentration of pups

Group code

0

125

250

500

T4 concentration

(μg/%)

6.849 ± 1.579

6.743 ± 2.433

6.498 ± 1.437

5.748 ± 0.907

T3 concentration

 (ng/ml)

0.729 ± 0.096

0.672 ± 0.139

0.660 ± 0.123

0.624 ± 0.073

TSH concentration (ng/ml)

0.384 ± 0.169

0.560 ± 0.312

0.417 ± 0.124

0.503 ± 0.146

Table No. 53: Weight of thyroid gland of pups

Pups

Group code

0

125

250

500

Male

0.0104

0.0103

0.0103

0.0102

Female

0.0103

0.0101

0.0103

0.0103

Table No. 54: Reproduction data

Reproduction data

Observed parameters

Group code

0µ

125

250µ

500

 Pairs started (N)

12

12

11

12

 Females showing evidence of copulation (N)

12

12

9µ©

12

 Females achieving pregnancy (N)

8

8

8

9

 Females with abortion (N)

0

0

0

0

 Conceiving days (duration of mating) 1 – 5 (N)

10

12

9

11

 Conceiving days (duration of mating) 6 – 10 (N)

2

0

0

0

Conceiving days (duration of mating) 11 – 14 (N)

0

0

2©

1

 Mean duration of mating (days)

2.17

1.42

5.27

3.08

 Pregnancy ≤ 21 days (N)

2

4

2

2

 Pregnancy = 22 days (N)

3

2

4

7

 Pregnancy ≥ 23 days (N)

3µ

2

2

0

 Mean duration of pregnancy (days)

22.25

21.25

22.00

21.78

 Females with live pups born (N)

6

8

8

9

 Females with live pups at day 4 after parturition (N)

6

8

7

9

 Number of implantations (mean)*

12.14

13.25

13.75

16.67

 Number of live born pups - at birth (mean)

10.14

12.13

11.50

14.56

 Number of live pups - at day 4 after parturition (mean)

10.00

12.00

10.88

14.44

 Number of live pups - at day 13 after parturition (mean)**

8.86

10.25

9.38

12.33

 Number of stillborn pups (total number/mean)

1/0.14

0/0

0/0

0/0

 Sex ratio (M/F) at birth (mean)

5.57 / 4.57

6.13 / 6.00

5.63 / 5.88

6.33 / 8.22

 Sex ratio (M/F) at day 4 after parturition (mean)

5.57 / 4.43

6.13 / 5.88

5.38 / 5.50

6.22 / 8.22

 Sex ratio (M/F) at day 13 after parturition (mean)

5.00 / 3.86

5.25 / 5.00

4.63 / 4.75

5.22 / 7.11

 Mean litter weight at birth (g)

70.74

83.60

78.01

97.98

 Mean litter weight at day 4 after parturition (g)

126.98

126.01

129.34

151.86

 Mean litter weight at day 13 after parturition (g)

310.50

284.24

299.71

342.72

Mean pup weight at birth (g)

6.58

7.05

6.62

6.77

Mean pup weight at day 4 after parturition (g)

10.93

10.42

10.22

10.61

Mean pup weight at day 13 after parturition (g)

31.46

28.90

27.63

28.39

Pup weight (g) at the time of AGD measurement (mean males/mean females)

11.09 / 10.63

10.62 / 10.19

10.55 / 10.35

11.02 / 10.36

Pup AGD (mm) on PND4 (mean males/mean females)

4.06 / 2.69

3.88 / 2.48

3.74/ 2.47

3.80/ 2.58

Corrected AGD (mean males/mean females)

1.82 / 1.23

1.77 / 1.14

1.73 / 1.15

1.71/ 1.18

Male pup nipple retention at day 13

0

0

0

0

ABNORMAL PUPS(with macroscopical findings)

 

Pups were not examined due to cannibalism or death

1

0

5

1

Autolysis of organs of pups

0

1

0

0

Mother with 0 (N)

6

7

7

8

Mothers with 1 (N)

0

1

0

1

Mothers with ≥ 2 (N)

0

0

0

0

Note:(N)–number of animals, PND – postnatal day, AGD – anogenital distance

* - calculated in pregnant females

** - number of live pups at day 13 was artificially decreased due to ofsacrificing of pups on PND4 (postnatal day 4) for T4 hormone assessment

µ- one female died

©- two females without sperm  

grey field= values statistically significant (p ≤ 0.05)

Table No. 55: Fertility parameters

Calculated parameters

Group code

0

125

250

500

 Male mating index

100.00

100.00

81.81

100.00

 Female mating index

100.00

100.00

81.81

100.00

 Male fertility index

66.67

66.67

72.72

75.00

 Female fertility index

66.67

66.67

72.72

75.00

 Gestation index

75.00

100.00

100.00

100.00

 Viability index on PND4

98.59

98.97

94.56

99.24

 LOSS OF OFFSPRING

 

 Post-implantation (implants minus live births) - mean

1.86

1.13

2.25

2.11

 Pregnant females with 0 (N)

3

4

0

3

 Pregnant females with 1 (N)

1

0

2

2

 Pregnant females with 2 (N)

1

3

3

1

 Pregnant females with ≥ 3 (N)

2

1

3

3

 Post-natal (live births minus alive at day 4) – mean

0.29

0.13

0.63

0.22
Conclusions:
NOAEL reproduction = 500 mg/kg bw/day
NOAEL developmental = 500 mg/kg bw/day
Executive summary:

The test item, benzenesulphonic acid, was tested for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test following OECD 422. The administration period was extended to 90 days. All findings of treated groups of animals were compared to the control group of animals.

During the study one female (at the control group) died for delivery complications. One male (at the highest dose level) and one female (at the mid dose level) died during the study due to misdoing error. These deaths were not related with the test item administration.

Decreased body weight in males at the highest dose level was recorded during the application period. In females at the highest dose level increased body weight at the 20th day of pregnancy was recorded. No effect of the test item on the food consumption was recorded in both sexes.

During the biometry of reproductive organs, statistically significantly decreased absolute weight of prostate gland with seminal vesicles in males at the mid and highest dose (reversible) levels were observed. In females, statistically significantly decreased absolute weight of pituitary gland at all dose levels was recorded. No findings related with the test item treatment were detected during the histopathological examination of reproductive organs and pituitary gland in animals at the highest dose level. All changed weights of reproductive organs in treated animals have no biological or toxicological significance.Examination of sperm motility and sperm morphology did not show any significant changes in males.

No findings related with the test item treatment were detected during the examination of microscopical structure of reproductive organs in males and femalesat the highest dose level. No effects on sperm motility and morphology in males.

No adverse changes in health condition and no clinical symptoms of intoxication were observed in animals following administration of the test item at any dose. Concentration of thyroid hormones and the weight of thyroid gland was similar to the control group. Number of females showing evidence of copulation was decreased at the mid dose level. In two females from this group, presence of sperm was not detected during the mating period. Mean duration of mating (days) was increased at the mid dose level. Number of treated females achieving pregnancy was similar in comparison with the control females. Stillborn pups were detected in one mother from the control group (one pup).

Cannibalism of pups in female was observed in the control group (one pup from one mother), at the mid dose level (5 pups from three mothers) and at the highest dose level (one pup from one mother).

Male and female mating index was decreased at the mid dose level, the fertility index was increased at the mid and highest dose levels. The gestation index was increased in females at the lowest, mid and highest dose levels in comparison with the control. The post-implantation and post-natal losses were not significantly changed in treated and control groups. The total number of pups at first litter check after parturition and during the next intervals were increased in all treated group of females. The mean number of pups per litter at first litter check after parturition and during the next intervals were increased in females at the highest dose level. The mean weight of litters was increased at the highest dose level at all examination intervals. The body weight of pups was decreased in all treated groups at the 12th and 13th day of the lactation period.

No presence of nipples in male pups was recorded. Statistically significant decrease of mean of anogenital distance in male pups at the mid and highest dose levels was recorded (without dose dependence). In male pups at the highest dose level statistically significantly decreased corrected anogenital distance was recorded. Examination of concentration of T4, T3 and TSH hormones and histopathological examination of thyroid gland no showed significantly changes, therefore decreased of the corrected anogenital distance was not considered to be of biological or toxicological significance. 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The database consists of reliable GLP studies.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The ASA Category comprises the following 5 aromatic sulphonic acids:


TSA, Toluene-4-sulphonic acid (EC 203-180-0, CAS 104-15-4)


XSA, (Xylenes and 4-ethylbenzene) sulphonic acid (EC 701-247-3, CAS -) Former EC 246-839-8


CSA, p-cumene sulphonic acid (EC 240-210-1, CAS 16066-35-6)


BSA, Benzene sulphonic acid (EC 202-638-7, CAS 98-11-3)


HBSA, 4-hydroxybenzensulphonic acid (EC No. 202-691-6, CAS No. 98-67-9)


 


The available results on developmental toxicity are:  
BSA, OECD 422 extended to 90 days (2020): NOAEL= 500 mg a.i./kg bw                     
HBSA, OECD 414 (2020): NOAEL = 1000 mg a.i./kg bw
SCS, OECD 414 (2020): NOAEL = 1000 mg a.i./kg bw  
CaXS, OECD 414 (1994): NOAEL = 936 mg a.i./kg bw  


SXS, OECD 414 (rabbits): NOAEL = 1000 mg a.i./kg bw


SXS, OECD 443 (2021): NOAEL = XX mg a.i./kg/bw


 


In OECD 414 on HBSA, the NOAEL for maternal toxicity and prenatal development was established as 1000 mg of a.i./kg bw/day. This NOAEL is based on no mortality of females, no changes in health condition status, no alteration to thyroid hormone measurements, no pathological findings in the dams and no dose-related changes in reproduction parameters and based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats dosed up to 1000 mg of a.i./kg bw/day.


BSA was tested under OECD 422 extended to 90 days toxicity and the the NOAEL for developmental toxicity was established as 500 mg of active ingredient /kg body weight/day which is the highest tested dose. This highest dose was selected based on the effects on the repeated dose part of the study. All changes in development parameters of pups at all dose levels were considered to be of no toxicological significance.


There are available studies for the chemically related hydrotrope substances that looked at reproductive organs. Hydrotropes are the salt form of the sulphonic acids and therefore are used as a support for this endpoint.


The study with CaXS performed under similar condition of OECD 414 guideline showed no adverse effects. The NOAEL for both maternal and foetal toxicity was the highest dose tested and the conclusion of the study was no indications of developmental toxicity including teratogenesis. The study performed on SCS confirms the absence of effects on developmental toxicity.


The 90-day oral rat and oral mouse studies and the 2-year chronic dermal rat and mouse studies on SXS included examination of sex organs of both sexes. No treatment related effects on developmental toxicity were reported at doses. A new OECD 443 was performed on SXS which is the most representative member of the hydrotropes category since it contains a significant proportion of both mono-alkyl (ethyl) and di-alkyl (dimethyl) chemical species and thus represents both the mono-alkyl and di-alkyl substances contained within the category.


 


The NOAEL value used for the risk assessment for developmental toxicity is 500 mg/kg bw from the OECD 422 extended to 90 days toxicity performed on BSA.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The database consists of reliable GLP studies.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

From the available studies of both aromatic sulphonic acid and related salts, there is no concerns for reprodution and developmenatl toxicity.


No classification for STOT is warrented at present under Regulation 1272/2008.

Additional information