Potassium 3-sulphonatopropyl acrylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Feb - 27 Mar 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, presentation of data in written and graphical format is limited. Based on the available data, the NOAEL derived by the study director was changed from 300 mg/kg bw/day to 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bureau For Chemical Substances, Poland

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium 3-sulphonatopropyl acrylate

EC Number:

250-465-0

EC Name:

Potassium 3-sulphonatopropyl acrylate

Cas Number:

31098-20-1

Molecular formula:

C6H10O5S.K

IUPAC Name:

potassium 3-(prop-2-enoyloxy)propane-1-sulfonate

Details on test material:

- Name of test material (as cited in study report): Ralu®Mer SPA (potassium-3-sulfonatopropyl acrylate)
- Physical state: white powder
- Analytical purity: 97.6%
- Batch No.: 05140043/003

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University, Białystok, Poland
- Age at study initiation: approximately 11 weeks old
- Weight at study initiation: males: 346.2-367.1 g, females: 210.2-217.8 g
- Housing: The animals were kept in plastic cages covered with wire bar lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). During the experiment, there were 3 or 5 animals/cage. Each sex was kept separately. For the purpose of mating, one female and one male were placed together. Pregnant females were housed individually. After delivery, females were housed with their offspring.
- Diet: Murigran standard granulated laboratory fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz, Poland; ad libitum
- Water: tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 35-48
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item were prepared daily (directly before the administration).

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle: 0.5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of the dose level of the test item were conducted by BLIRT S.A. Laboratory, Trzy Lipy 3/1.38, 80-172 Gdańsk, Poland.
Samples (aqueous solutions of the dose levels) collected on each treatment day were frozen (ca. - 20 °C) and sent to BLIRT S.A. (143 samples in four batches). The samples stored in a freezer (at -20 ± 5 °C) were stable for at least 25 days.
The dosel levels in the samples were within the following ranges:
18.815 - 20.533 mg/mL for the dose level of 20 mg/mL (94.1 – 102.7%)
59.104 - 62.994 mg/mL for the dose level of 60 mg/mL (98.5 – 105.0 %)
199.265 - 220.722 mg/mL for the dose level of 200 mg/mL (99.6 – 110.4%)
The results were within the range of 80 -120%.

Duration of treatment / exposure:

males: 28 days
females: 43-49 days (2 weeks prior to mating; variable time to conception; duration of pregnancy; until 4 days after delivery)
satellite groups (males and females): 28 days (observation for 14 days post-treatment)

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 12 females per dose
5 males and 5 females per satellite group (vehicle satellite control group and 1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the outcome of a 7-day dose range finding study, the dose levels of 100, 300, and 1000 mg/kg bw/day were selected for the main study.
- Post-exposure recovery period in satellite groups: 14 days
Two satellite groups of five animals per sex in the control and the high dose group were used for observation of reversibility, persistence or delayed occurrence of systemic toxic effects. The satellite groups were treated for 28 days and observed for 14 days post-treatment. The animals of the satellite groups were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (once a day on weekends and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day (once a day on weekends and public holidays)

BODY WEIGHT: Yes
- Time schedule for examinations: males: twice per week; females: twice per week during pre-mating; on Days 0, 7, 14 and 20 of gestation; on Day 0 or Day 1 post-partum and on Day 4 post-partum
Body weights of pups were measured on Day 0 or Day 1 post-partum and on Day 4 post-partum.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content
weights of internal organs: testes, epididymides and ovaries (of all surviving animals); brain, thymus, heart, liver, spleen, kidneys, and adrenal glands of 5 adult males and 5 adult females from each group

HISTOPATHOLOGY: Yes
The following organs and tissues were examined: brain with the cerebellum, spinal cord, muscle with the peripheral nerve, salivary glands, lymph nodes, trachea, thyroid with the parathyroids, stomach, duodenum, jejunum, ileum, caecum, colon, liver, pancreas, spleen, lungs, heart, thymus, kidneys, adrenal glands, urinary bladder, ovaries, uterus with the cervix, testicles, epididymides, accessory sex glands (prostate with the seminal vesicles and coagulating glands), and organs showing gross lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on Day 4 post-partum
- These animals were subjected to postmortem examinations

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Student’s t-test (p ≤ 0.05); one-way analysis of variance; Dunnett’s test (p ≤ 0.05)

Reproductive indices:

Fertility of parental animals:
Mating index for males:
the number of males showing evidence of copulation/the total number of mated males x 100
Mating index for females:
the number of sperm-positive females/the total number of mated females x 100
Fertility index for males:
the number of males that impregnated females/the total number of mated males x 100
Fertility index for females:
the number of pregnant females/the total number of sperm-positive females x 100
Pregnancy index:
the number of litters with live births/the number of pregnant females x 100

Offspring viability indices:

Index of live births:
the total number of live newborns/the total number of newborns x 100
Index of 4-day survival:
the number of live sucklings after 4 days/the number of live newborns x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two males from the low dose group and two females from the high dose group died during the treatment period (not treatment-related)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in the average body weight in high dose females on gestation Day 20 (non-adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in the average body weight in high dose females on gestation Day 20 (non-adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lesions were observed in several tissues; however, none of the lesions observed had a specific character or showed severity with increasing dose level (non-adverse)

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Two males from the low dose group (Day 25 and Day 28) and two females from the high dose group (Day 5 and Day 39) died during the treatment period. In both males and in the female that died on Day 39 perforation of the esophagus was found, indicating a gavage error. These deaths were not considered to be treatment-related. In the second female that died no abnormalities were found at necropsy and the death was considered to be incidental.
Decreased locomotor activity, a decreased rate of reaction to sound, and bleeding from the snout were observed in one male in the low dose group (the animal was found dead on Day 28). Alopecia and a scab on the snout were noted in one male from the mid dose group (temporary changes). There were no other abnormal differences in appearance and behaviour between the treated and the control groups. Animals in the satellite groups showed no delayed occurrence of adverse effects.

BODY WEIGHT AND WEIGHT GAIN
During the pre-mating and post-mating period, there were no statistically significant differences in average male body weights between the treated and the control groups.
In females of the high dose group, the average body weight was statistically significantly decreased on gestation Day 20 compared to the control group. However, the body weights were found to be comparable with control group in the lactation phase. As body weights of the high dose females were only decreased by 8.1% (control females: 340.1 ± 22.91 g; high dose females (1000 mg/kg bw/day): 312.7 ± 27.44 g), this finding was not considered to be toxicologically relevant. According to Chou et al. (1998), body weight loss or decrease in body weight gain of less than 10% is considered non-adverse. In females of the other dose groups, there were no statistically significant differences in average body weights between the treated and the control groups during the pre-mating, gestation and lactation period. Animals in the satellite group showed no statistically significant differences in body weights of males and females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All the females and males were mated (see Table 1). The average number of days needed for copulation was 1.5 in the control group, 1.2 in low dose group, 2 in the mid dose group and 1.4 in the high dose group. All the females (12/12) were pregnant in the control, low and mid dose groups and all the females in these groups delivered live offspring. In the high dose group, only 11 females were mated as one female was died on day 5 of the experimental phase. One high dose female was not pregnant and another female died during the gestation phase. Therefore, only 9/11 females in the high dose group delivered offspring.
All the females from the control and treatment groups delivered live offspring. The indices referring to fertility of the parental animals and the length of gestation are presented in Table 2. There were no significant differences in the fertility parameters between the control and treatment groups.

ORGAN WEIGHTS
The analysis of absolute and relative weights of internal organs of the animals from the high dose group showed statistically significant changes compared with the control group. A decrease in absolute and relative weights of the thymus and an increase in absolute and relative weights of the spleen in high dose males were found. In high dose females, an increase in absolute and relative weights of the spleen, and in relative weights of the kidneys were observed. In males and females from the low and mid dose groups, no statistically significant changes were noted compared to the control group. The analysis of absolute and relative weights of the gonads showed a statistically significant increase in absolute weights of the ovaries in females in the low dose group.
Males in the satellite group showed statistically significantly decreased thymus weights (absolute) and statistically significantly increased epididymides weights (relative). In females in the satellite group a statistically significant increase in absolute and relative weights of the kidneys and a statistically significant decrease in relative weights of the brain were observed. No other statistically significant changes in absolute and relative weights of the remaining organs were noted in the satellite group.
No macroscopic or microscopic abnormalities were found in these organs during the gross pathology and histopathological examinations. Furthermore, the toxicokinetic behaviour of the test substance gives no indication that any of the organs with changed weights are target organs of the test substance. According to the Ecetoc technical report No. 85 (2002), increased organs weights with no evidence of pathological and histopathological changes should be considered to be a non-specific response (i.e. not adverse).
It is common understanding that alterations in organ weights are not necessarily toxicologically relevant if there are no other indications of organ-specific adverse effect and should therefore not be considered as an adverse effect when no macroscopic abnormalities and no histopathological findings were observed for in the organs. Thus, the observed effects were not considered to be treatment-related.

GROSS PATHOLOGY
Macroscopic changes of parental animals that died during the study period:
In the low dose group two males died during the study period. In both males perforation of the esophagus was observed indicating gavage error. The presence of clear fluid in the pleural cavity of the lungs and swelling of the right part of the body, i.e. the cervical and the scapular parts were found.
In the high dose group two females died during the study period. In one female esophageal perforation and the presence of clear fluid in the thoracic cavity was noted. In the second female no abnormalities were found.
Macroscopic changes of parental animals euthanized at the end of the treatment period:
In the control group, an inflammatory focus around the left ventricle of the heart in one male and paleness of the liver and a lesion in the left liver lobe in one female were observed. There were no other gross changes in any other dose group. There were no macroscopic changes in the organs and tissues of the rats in the satellite group.

HISTOPATHOLOGY:
The histopathological examination of the parental animals that died during the treatment period revealed lesions associated with perforation of the esophagus and damage to surrounding tissues (circulatory disorder- hyperemia of the salivary glands, mandibular lymph nodes, thymus, lungs, and heart, as well as emphysema in the lungs), indicating a gavage error.
The histopathological examination of organs and tissues of the euthanized parental animals at the end of the study revealed a few changes in individual animals. Lesions were observed in the brain, cerebellum, lungs, heart, liver, spleen, pancreas, intestines (duodenum, jejunum, cecum, and colon), kidneys, adrenals, and prostate. The histopathological changes observed in these organs were associated with various types of disorders. However, none of the lesions observed in the treated animals had a specific character or showed severity with increasing dose levels. Most of them occurred in the control and the treated groups at the same time with the same incidence. Some of them were observed only in the control group.
The histopathological examination of organs and tissues of the euthanized animals in the satellite group revealed a few changes in individual animals. Lesions were observed in the cerebellum, heart, liver, adrenals and prostate. The histopathological changes observed in these organs were associated with various types of disorders. However, none of the lesions observed in the treated animals had a specific character or showed severity with increasing dose level.
Therefore, the observed effects were not considered to be treatment-related

References:
ECETOC Technical Report 85, 2002, Recognition of, and differentiation between, adverse and non-adverse effects in toxicological studies, ISSN-0773-6347-85
Chou et al., 1998, Minimal risk level (MRLs) for hazardous substances. J. Clean Technol., Environ. Toxicol., & Occup. Med., Vol. 7, No. 1, 1998
Derelanko, M.J., 2008, The toxicologist´s pocket handbook. 2nd edition, informa healthcare, ISBN 987-142005138-4

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponding to the highest dose tested

2

Dose descriptor:

NOAEL

Remarks:

toxicity to reproduction

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponding to the highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

temporary hematoma were found on the snout, back, head, abdomen; non adverse

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

stillborn pups, cannibalism, One male pup from the high dose group died on day 1 post-partum; not a treatment-related effect

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

average body weight of pups/litter in the high dose group was statistically significantly decreased on day 0 and 4 post-partum compared to the control group; less serious adverse effect and thus not considered to be toxicologically relevant

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There were 559 pups. Six of them were stillborn (2 females in the control group, 3 males in the mid dose group, and 1 male in the high dose group). One male pup from the high dose group died on Day 1 post-partum.
Nine pups were cannibalised by mothers between Day 0 and Day 4 post-partum (one female in the control group, one male and one female in the low dose group 1, one male and 3 females in the mid dose group, and 2 females in the high dose group).
The effects were not considered to be treatment-related.
The average numbers of pups per litter are presented in Table 3. No statistically significant differences were observed between the treatment groups and the control group regarding the number of pups per litter and sex per litter.
The indices relating to offspring survival are presented in Table 4. There were no significant differences in the offspring parameters between the control and treatment groups.

CLINICAL SIGNS (OFFSPRING)
The following observations were made in offspring:
- temporary hematoma on the snout in two males from the control group, in one male from the low dose group, in one male from mid dose group, and one female from high dose group;
- temporary hematoma on the back in two females from control group and in one male from the mid dose group;
- temporary hematoma on the snout and on the back in one male from the control group
- temporary hematoma on the snout and on the head in one male from mid dose group;
- hematoma on the back in one female from high dose group;
- temporary hematoma on the head in one male from the control group and in one female from the mid dose group;
- temporary hematoma on the abdomen in one female from the high dose group;
- livid snout in one female from low dose group and one female from mid dose group;
- livid body in one female from the high dose group.
The observed clinical signs were not considered to be treatment-related as no dose-response relationship was noted.

BODY WEIGHT (OFFSPRING)
Average body weights of the pups are shown in Table 5.
There were no statistically significant changes in average body weights of the pups per litter in the low and mid dose groups compared to the control group on the day of parturition (Day 0) and on Day 4 post-partum. For the high dose group, the average body weight of pups per litter was statistically significantly decreased on (Day 0) and on Day 4 post-partum compared to the control group. As body weight loss or decrease in body weight gain of 10-19% is considered as a less serious adverse effect (Chou et al., 1998), the effect was not considered to be toxicologically relevant taking into account all available data on offspring.

GROSS PATHOLOGY (OFFSPRING)
There were no treatement-related macroscopic lesions or malformations in the pups found dead and in the pups euthanized on Day 4 post-partum.

Reference
Chou et al., 1998, Minimal risk level (MRLs) for hazardous substances. J. Clean Technol., Environ. Toxicol., & Occup. Med., Vol. 7, No. 1, 1998

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponding to the highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Mating of parental animals

Parameter	GROUP [mg/kg bw/day]
	0	100	300	1000
Number of males to bemated	10	10	10	10
Number of females to be mated	12	12	12	111
Number of males which copulated	10	10	10	10
Number of females which were mated	12	12	12	11
Average number of days to copulation	1.5	1.2	1.2	1.4
Number of fertilized females	12	12	12	11
Number of pregnant females	12	12	12	10
Number of non-preganant females	0	0	0	1
Number of females which delivered offspring	12	12	12	92
Number of females which delivered live offspring	12	12	12	9

1 – one female [22] died during the 1st week of the experiment

2 – one female [17] died during the gestation period

 

 Table 2: Indices relating to fertility of parental animals

Parameter	GROUP [mg/kg bw/day]
	0	100	300	1000
Mating index for males	100.0	100.0	100.0	100.0
Mating index for females	100.0	100.0	100.0	100.0
Fertility index for males	100.0	100.0	100.0	90.9
Fertility index for females	100.0	100.0	100.0	90.9
Pregnancy index	100.0	100.0	100.0	100.01
Length of gestation	22.1 ± 0.3	22.2 ± 0.4	22.2 ± 0.4	21.9 ± 0.6

1–excluding the femalewhich died duringthe gestation period

 

Table 3: Average number of pups per litter

Parameter	GROUP [mg/kg bw/day]
	0	100	300	1000
Total number of pups per litter	13.2 ± 2.1	12.4 ± 2.1	12.3 ± 2.6	11.7 ± 1.5
Number of live births per litter	13.0 ± 2.2	12.4 ± 2.1	12.0 ± 2.4	11.6 ± 1.5
Number of pups per litter, min.–max.	9-17	10-16	8-17	9-14
Percentage of males per litter	51.9	48.3	39.5	53.3
Percentage of females per litter	48.1	51.7	60.5	46.7

 

Table 4: Indices relating to survival of offspring

Parameter	GROUP [mg/kg bw/day]
	0	100	300	1000
Index of live births	98.7	100.0	98.0	99.0
Index of 4-day survival	99.4	98.7	97.2	97.1
Mortality [%]	0.6	1.3	2.8	2.9

 

Table 5: Average body weights [g] of pups/litter on Days 0 and 4 post-partum

Day	Group [mg/kg bw/day]
	0 n=12	100 n=12	300 n=12	1000 n=9
0	76.6  ±  9.23	73.7  ±  12.30	71.3  ±  12.38	63.2  ±  5.20*
4	112.8 ± 14.30	109.5 ±  15.34	106.1 ±  16.85	96.0  ± 12.69*

n –number of dams per group; mean± SD; *-statistically significant difference at p ≤ 0.05

 

Applicant's summary and conclusion

Conclusions:

Based on the results of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL for reproduction and developmental toxicity was considered to be ≥ 1000 mg/kg bw/day.