Potassium 3-sulphopropyl methacrylate

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:

guideline study

Remarks:

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.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see target record

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

target record

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

19 Feb - 27 Mar 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Klimisch 1 source record, but performed on read-across substance

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rational for the analogue approach is the high structural similarity between the source and the target substance. 2-Propenoic acid, 3-sulfopropyl ester, potassium salt (source) and 2-Propenoic acid, 2-methyl-,3-sulfopropylester, potassium salt (target) are structurally identical except an additional methyl group on position 2 of the target substance. Despite the fact that a methyl group may alter the toxicological behaviour of a substance, this effect is considered very minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the ester and the Michael-acceptor system. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: 2-Propenoic acid, 3-sulfopropyl ester, potassium salt, CAS 31098-20-1, EC 250-465-0, SMILES [K+].[O-]S(=O)(=O)CCCOC(=O)C=C, MW 232.30

Target Chemical: 2-Propenoic acid, 2-methyl-,3-sulfopropylester, potassium salt, CAS 31098-21-2, EC 250-466-6, SMILES [K+].CC(=C)C(=O)OCCCS(=O)(=O)O, MW 247.33

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional methyl group of the target chemical becomes obvious. So the molecular weight is in the same range, i.e. approx. 240 g/mol, indicating per se the potential for absorption. Both substances are solids which either do not melt or decompose at or above 300°C, and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range, < -3.
In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria or chromosome aberrations in mammalian cells, both substances are not genotoxic. With regard to gene mutation in mammalian cells, SPM was tested negative ± S9, SPA however positive ± S9, although 2-fold increases over background were noted predominantly in cytotoxic concentrations, no differences in the ratio small/large colonies were observed. So it may be concluded that SPM is by trend even less harmful than SPA, indicating that read-across may represent a worst-case scenario and does not underestimate the actual hazard of SPM.
In summary, the target chemical 2-Propenoic acid, 2-methyl-,3-sulfopropylester, potassium salt, does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint skin sensitization:

Endpoint Source: SPA Target: SPM
Molecular weight 232.30 g/mol 247.33 g/mol
Physical state solid solid
Partition coefficient logPow = -3.63 logPow = -3.1 (EpiSuite estimation)
Water solubility 3329 g/L 2570 g/l
Acute toxicity oral LD50 > 5000 mg/kg (rat) LD50 > 5000 mg/kg (rat)
Acute toxicity dermal LD50 > 2000 mg/kg (rat) n/a
Skin irritation Not irritating (in vivo, rabbit) Not irritating (in vivo, rabbit)
Eye irritation Not irritating (in vivo, rabbit) Not irritating (in vivo, rabbit)
Skin sensitization Skin sens 1A (GPMT) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) Negative ± S9 (OECD 471; two independent studies)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 473) Negative ± S9 (OECD 473)
Gene mutation in mammalian cells Positive ± S9 (OECD 476) Negative ± S9(OECD 476)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a

Reason / purpose for cross-reference:

read-across source

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

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

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 dose 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

Remarks:

Doses / Concentrations:100, 300, 1000 mg/kg bw/dayBasis:actual ingested

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.

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

Clinical signs:

effects observed, non-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)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

Key result

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

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

Remarks on result:

not determinable due to absence of adverse toxic effects

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, non-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

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 treatment-related macroscopic lesions or malformations in the pups found dead and in the pups euthanized on Day 4 post-partum.ReferenceChou et al., 1998, Minimal risk level (MRLs) for hazardous substances. J. Clean Technol., Environ. Toxicol., & Occup. Med., Vol. 7, No. 1, 1998

Key result

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Conclusions:

The study was conducted under GLP according to OECD guideline 422. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats, as an OCED 422 study both covers repeated dose and reproductive (screening) toxicity testing.
In general is should be mentioned ahead that study duration, although not yet subchronic, is prolonged compared to an OECD 407 study (28 days (males) and ca. 43-49 days (females) compared to 28 days). Also, females are pregnant, making them in general more susceptible for toxic effects compared to non-pregnant animals. Hence, testing via an OECD 422 may reveal more distinct effects than testing via an OECD 407, and the observed effects may count as worst case short-term toxicity testing.
As described above, the observed deaths were considered to be either due to a gavage error or incidental, also, no dose-related clinical signs were noted.
The analysis of body weights showed no effects in males, only a decrease in body weights of females (group 3) on day 20 of gestation. 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) (Chou et al., 1998, Minimal risk level (MRLs) for hazardous substances. J. Clean Technol., Environ. Toxicol., & Occup. Med., Vol. 7, No. 1, 1998), body weight loss or decrease in body weight gain of less than 10% is considered non-adverse.
The ophthalmic examinations did not reveal any pathological changes.
A statistically significant decrease in the number of erythrocytes and a statistically significant increase in MCV and MCH levels were observed in males and females in the high dose group compared with the control group. Furthermore, the number of reticulocytes in males from the high dose group was statistically significantly increased. No other hematological parameters showed any significant differences between the treated and the control groups.
Considering various historical control data for hematological parameters for Wistar (CRL:WI) rats (published by Charles River Laboratories) the data indicate that the statistically significant differences observed for MCV, MCH, erythrocytes and reticulocytes in the high dose group are within or similar to the normal biological variation of Wistar rats.In addition, the differences in hematological parameters for the Wistar rats are within or similar to the range of historical control values of CD® rats and F-344 rats (Derelanko, 2008: Derelanko, M.J., 2008, The toxicologist´s pocket handbook. 2nd edition, ISBN 987-142005138-4).Therefore, it can be considered that the differences in hematological parameters in male and female Wistar rats, although statistically significant, are unlikely to be treatment-related effects and thus non-adverse. Also, effects in the erythrocyte/leukocyte system were considered to be non-adverse taking into account information from the satellite groups.
No treatment-related findings resp. findings with no dose-response in biochemical and enzymatic determinations were observed.
No significant changes were observed in the urinalysis parameters except an increase in the number of leukocytes in females from the high dose group. As no dose-response relationship was noted and the effect was not observed in the satellite group, the finding was considered to be non-adverse.
In open field observations, there were no dose-related or non-incidental effects noted. There were no treatment-related effects on responses to visual, tactile, auditory, and pain stimuli. There were no statistically significant differences in the fore- and hindlimb grip strength of males and females in the satellite group, indicating that the observed effect on forelimb strength in high dose males was incidental. No effects were observed for other treated males and females.
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. However, 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.
There were no macroscopic changes in the organs and tissues considered to be dose-related or non-incidental.
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. Therefore, the observed effects were not considered to be treatment-related.

With regard to reproductive performance, all the females and males were mated. 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. There were no significant differences in the fertility parameters between the control and treatment groups.
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. The effects were not considered to be treatment-related. 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. There were no significant differences in the offspring parameters between the control and treatment groups.
The observed clinical signs were not considered to be treatment-related as no dose-response relationship was noted.
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.
There were no treatment-related macroscopic lesions or malformations in the pups found dead and in the pups euthanized on Day 4 post-partum.

In conclusion, the treatment of male and female rats with SPA at the dose levels of 100 and 300 mg/kg bw/day by oral gavage revealed no or no adverse effects. All observed effects of the high dose group were also considered to be incidental or non-adverse, e.g. due to an adaptive response. Based on the results of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of SPA in rats, the no observed adverse effect level (NOAEL) of the test item was determined to be 1000 mg/kg b.w./day for parental, reproduction, and developmental toxicity.

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, “Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.
Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”
There were no significant differences in the fertility parameters between the control and treatment groups, nor in the offspring parameters between the control and treatment groups, nor no treatment-related macroscopic lesions or malformations in the pups found dead and in the pups euthanized on Day 4 post-partum, so no classification as reproductive or developmental toxicant is triggered.

Executive summary:

The objective of this study was to determine and evaluate potential toxicity of potassium-3-sulphonatopropyl acrylate (SPA) following oral administration to rats in a OECD 422 study under GLP. The study involved the determination of the effects of the test item on body weights, food intake, behaviour, clinical, hematological, coagulation, biochemical, enzymatic, urine parameters and the evaluation of gross and histopathological lesions in tissues and internal organs. The study also provided initial information on the effects on reproductive performance of males and females and the development of offspring.

The study was divided into two stages, the dose range-finding study (DRF study) and main study

 

Dose range-finding study (DRF study)

The aim of the DRF study was to select doses for the main study.

It was performed on 20 male and 20 female Wistar rats (Cmdb:WI; outbred).

The animals in the DRF study were divided into four groups. These were:

● one control group (0) which was given distilled water (vehicle control),

● three groups exposed to SPA, i.e.:

· group 1 given SPA at the dose of 100 mg/kg b.w./day

· group 2 given SPA at the dose of 300 mg/kg b.w./day

· group 3 given SPA at the dose of 1000 mg/kg b.w./day

There were 5 males and 5 females in each group.

The test item/ vehicle control was administered every day for 7 days.

 

Course of the DRF study:

Clinical studies: During the experiment, mortality, morbidity, body weights, and food intake were recorded. After 7 days of the experiment, the animals were anesthetized to collect blood for clinical-chemical examinations. Next, they were euthanized and subjected to post-mortem examinations.

Clinical-chemical examinations: After 7 days of the experiment, hematological examinations of peripheral blood, biochemical and enzymatic examinations of serum were conducted.

Post-mortem examinations: All animals used in the DRF study were subjected to these examinations. They involved the observation of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content.

 

Results of the DRF study:

Clinical studies: All animals survived the experiment. No clinical signs were observed. No statistically significant differences in body weights between the treated and the control groups were noticed. Food intake in the treated and the control groups was similar (no statistical analysis).

Clinical-chemical examinations: The hematological, biochemical, and enzymatic examinations did not reveal any statistically significant changes.

Post-mortem examinations: The macroscopic examinations of all animals used in the DRF study did not reveal any lesions.

 

DRF study summary:

Based on the results of the 7-day dose range finding study, the dose levels of 100, 300, and 1000 mg/kg b.w./day were selected for the main study.

 

Main study

The main study was performed on 50 male and 58 female Wistar rats (Cmdb:WI; outbred). The animals used in the main study were divided into four groups. There were 10 males and 12 females in each group. The groups included:

● one control group (0) which was given distilled water (vehicle control),

● group 1 given SPA at the dose of 100 mg/kg b.w./day,

● group 2 given SPA at the dose of 300 mg/kg b.w./day

● group 3 given SPA at the dose of 1000 mg/kg b.w./day

Furthermore, two satellite groups were used. These were:

● one control group (0SAT) which was given distilled water (vehicle control),

● one treated group (3SAT) given SPA at the dose level of 1000 mg/kg b.w./day

The satellite groups were treated for 28 days. After that, they were observed for 14 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects. There were 5 males and 5 females in each satellite group.

After two weeks of initial treatment in the main study, the animals were mated (one male/one or two females). The satellite groups were not mated. The test item/vehicle control was administered to males for 28 days. Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 4 days after delivery (43 – 49 days).

 

Course of the main study:

Clinical studies: During the main study, mortality, morbidity, body weights, and food intake were recorded. Moreover, ophthalmic examinations and behavioural studies were conducted. The latter involved observations of animal behaviour in an open field, responses to stimuli, locomotor activity, and fore- and hindlimb grip strength. 5 adult males and 5 adult females from each group were subjected to the behavioural studies.

The following parameters were determined in offspring: the number of pups in a litter, the numbers of live births and stillbirths, sex of pups, the weight of a litter. Furthermore, abnormalities in body structure and behaviour of pups as well as their mortality up to day 4 of life were recorded. After 28 days (males) or 42 days (SAT groups, males and females) of the experiment, the animals were anesthetized to collect blood for clinical-chemical examinations. Next, they were euthanized and subjected to post-mortem examinations. Pups were euthanized on day 4 after parturition, whereas females on the following day. The females were anesthetized to collect blood for clinical-chemical examinations. After euthanasia, the females and the pups were subjected to post-mortem examinations.

Clinical-chemical examinations: At the end of the experiment, 5 adult males and 5 adult females from each group were subjected to hematological examinations of peripheral blood and bone marrow, coagulation examinations, biochemical and enzymatic examinations of serum, general urinalyses, and examinations of urine sediment.

Post-mortem examinations: The animals used in the main study were subjected to the detailed gross examination which involved observations of the external body surface, all natural apertures and the cranial, thoracic, and abdominal cavities with their content. The numbers of implantation sites and corpora lutea in parental females used in the main study were determined. Absolute and relative weights of the testes, epididymides, and ovaries of all surviving adult animals were determined. Additionally, absolute and relative weights of the brain, thymus, heart, liver, spleen, kidneys, and adrenals of 5 adult males and 5 adult females from each group were determined. The histopathological examination of the following organs was conducted: brain with the cerebellum, spinal cord, muscle with the peripheral nerve, salivary glands, lymph nodes, trachea, thyroid with the parathyroids, stomach, duodenum, jejunum, ileum, cecum, colon, liver, pancreas, spleen, lungs, heart, thymus, kidneys, adrenals, urinary bladder, ovaries, uterus with the cervix, testicles, epididymides, accessory sex glands (prostate with the seminal vesicles and coagulating glands), and all organs showing gross lesions.

Evaluation of the immune system: An evaluation of the immune system of 5 adult males and 5 adult females was based on the results of clinical-chemical examinations and post-mortem examinations of the spleen, thymus, and lymph nodes, as well as the statistical analysis of the absolute and relative weights of the spleen and thymus. The clinical-chemical parameters included blood morphology with a picture of peripheral blood and bone-marrow smears, the concentrations of albumin, total protein, the albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase.

 

Results of the main study:

Clinical studies: During the experiment, mortality of the parental adults was observed. Four animals died during the main study (two males from group 1 and two females from group 3). As for offspring, 6 pups were born dead (2 females in the control group, 3 males in group 2, and 1 male in group 3). The remaining offspring (with the exception of nine pups eaten by mothers) survived the 4-day observation period. The only exception was 1 pup which died one day after the birth (1 female in group 3). These cases of mortality were not test item-related effects.

There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. The animals showed a few pathological clinical signs during the entire experiment. However, these were not test item-related effects. There were no differences in appearance and behaviour of pups between the treated and the control groups.

The analysis of body weights showed a decrease in body weights of females (group 3) on day 20 of gestation and a decrease in body weights of pups in a litter (group 3) on days 0 and 4 of life.

No treatment-related effects were observed during the detailed behavioural studies. On the basis of the detailed clinical observations, made during and after the treatment, and the open field observations, no muscarinic symptoms (weakness, lacrimation, salivation, and anxiety), nicotinic symptoms (diarrhea and motor coordination difficulties), or central symptoms were noticed. Delayed neurotoxicity, i.e. paralysis of the hindlimbs, clumsiness, sensory disturbances, and general paralysis were not observed. The evaluation of responses to stimuli showed no effects of the test item. On the basis of the fore- and hindlimb grip strength measurement, the detailed clinical observations, and the open field observations (body posture, gait, and locomotor activity), it can be concluded that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day (males and females) did not cause any miotoxic changes.

The indices relating to fertility of the parental animals, including mating indices for males and females, fertility indices for males and females, and pregnancy indices in the treated and the control groups were similar, which suggests that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day did not negatively influence fertility of the parental rats.

The test item did not negatively influence the total number of pups in a litter, the numbers of live births and stillbirths in a litter, and the percentages of males and females in a litter in groups 1 and 2.

It proves the lack of harmful effects of the test item at these doses on fetuses. However, the analysis of body weights showed a decrease in body weights of pups in a litter (group 3) on days 0 and 4 of life. The percentages of males and females in a litter in group 3 were similar. The indices relating to survival of offspring of the treated parents show no negative effects on the indices of live births and 4-day survival in comparison with the control group.

On the basis of the results of this study of SPA on rats, it may be concluded that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day (males and females) had no adverse effects, as clinical observations, and effects body weight and food intake measurements may not be considered as adverse.

On the basis of the results of the behavioural studies, it may be concluded that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day (males and females) did not affect the nervous system. Moreover, it did not cause miotoxic changes.

On the basis of pup body weight measurements and the results of the evaluation of reproduction, it may be concluded that the test item at the doses of 100 and 300 mg/kg b.w./day had no adverse effects. A decrease in body weights of pups in litters in group 3 (1000 mg/kg b.w./day) was observed.

Clinical-chemical examinations: Statistical analyses of the clinical-chemical examinations results showed a few statistically significant changes. Some of them were accidental, whereas the others should be perceived as caused by the test item.

Changes which can be perceived as caused by the test item are:

● a decrease in the number of erythrocytes in males and females from group 3,

● an increase in MCV and MCH in males and females from group 3,

● an increase in the number of reticulocytes in males from group 3.

● an increase in the number of polychromatophilic erythroblasts (bone marrow) in males from group 3,

● an increase in the number of orthochromatic erythroblasts (bone marrow) in males from group 3,

● an increase in the total number of cells in the erythrocyte system (bone marrow) in males from

group 3,

● a decrease in the total number of cells in the leukocyte system (bone marrow) in males from group 3,

● a decrease in the leukocyte system/erythrocyte system quantitative ratio (bone marrow) in males

from group 3.

Both coagulation and enzymatic examinations did not reveal any statistically significant changes. As for the biochemical and urine examinations, only accidental changes were stated. The hematological examinations of the satellite groups showed a statistically significant increase in MCV in females from group 3SAT. The increase after the break in the exposure indicated that the test item influence on this parameter was still observed. However, no treatment-related findings resp. findings with a dose-response in biochemical and enzymatic determinations were observed. The remaining clinical-chemical examinations of the satellite group did not reveal any other statistically significant changes.

On the basis of the clinical-chemical examinations, it can be stated that 28-day (males) or 43 – 49-day (females) exposure of rats to potassium-3-sulphonatopropyl acrylate (SPA) at the dose of 1000 mg/kg b.w./day may cause some hematological changes, but none which have to be considered adverse. Increased MCV in females from group 3SAT could be a stable sign. For the doses of 100 and 300 mg/kg b.w./day, no treatment-related changes were observed.

Post-mortem examinations: There were no macroscopic and histopathological changes caused by the test item in examined tissues and organs of the parental males and females from all groups, i.e. 0, 0SAT, 1, 2, 3, and 3 SAT.

On the basis of the results of the study and the statistical analysis of the numbers of corpora lutea and implantation sites in the ovaries of the parental females, it can be concluded that the test item did not cause any changes in the numbers of corpora lutea and implantation sites when compared to the control group. The macroscopic evaluation of the offspring did not reveal any lesions or malformations in live born and stillborn pups.

The analysis of absolute and relative weights of internal organs showed some statistically significant changes however only thymus and spleen absolute and relative weights in males in the high dose group were statistically significantly changed. For females in the high dose group, only spleen absolute and relative weights were statistically significantly increased. Alterations in organ weights are not toxicologically relevant They should not be considered as an adverse effect as no macroscopic abnormalities and no histopathological findings were observed in these organs.

The macroscopic and the histological examinations of the rats treated with potassium-3-sulphonatopropyl acrylate (SPA) at the doses of 100, 300, and 1000 mg/kg b.w./day did not reveal any toxic effects of the test item on reproductive organs and other tissues and organs of the adults. Increased organ weights with no evidence of pathological and histopathological changes should be considered as a non-specific response (i.e. not adverse). There were no macroscopic lesions and malformations in the offspring.

Evaluation of the immune system: The evaluation of the immune system involved a few types of examinations. The first one was the examination of peripheral blood morphology and bone marrow. For this study, some differences in the number of erythrocytes, MCV, MCH, and the number of reticulocytes were stated. A decrease in number of erythrocytes and an increase in MCV and MCH in males and females from group 3, an increase in the number of reticulocytes in males from group 3, and an increase in MCV in females in the satellite groups were noticed. For bone marrow, an increase in the number of polychromatophilic and orthochromatic erythroblasts as well as an increase in the total number of cells in the erythrocyte system in males from group 3 were stated. As for the bone marrow leukocyte system, a decrease in the total number of cells in the leukocyte system in males from group 3 and a decrease in the leukocyte system/erythrocyte system quantitative ratio in males from the group 3 were stated.

The next group of studies relating to the immune system were the assessments of several biochemical parameters such as the concentrations of albumin, total protein, albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of AST, ALT, and alkaline phosphatase. Evaluated as accidental the decreased albumin concentration and the albumin/globulin ratio in males from group 2 were stated. Other biochemical parameters did not change statistically significantly.

On the basis of the clinical-chemical parameters, no impact of the test item at the doses of 100 and 300 mg/kg b.w./day on the immune system was stated. For the dose of 1000 mg/kg b.w./day, it is difficult to state whether the changes were related to the hematological system or the immune system. The macroscopic and the histopathological examinations of the thymus, spleen, and lymph nodes did not reveal any harmful effects of the test item on the immune system. Only thymus and spleen absolute and relative weights in males in the high dose group were statistically significantly changed. For females in the high dose group, only spleen absolute and relative weights were statistically significantly increased. Alterations in organ weights are not toxicologically relevant. They should not be considered as an adverse effect as no macroscopic abnormalities and no histopathological findings were observed in these organs.

On the basis of the clinical-chemical parameters, the histopathological evaluation of the thymus, spleen, and lymph nodes, and the statistical analysis of absolute and relative weights of the thymus and spleen in groups 1 and 2, no impact of the test item on the immune system was stated. For the dose of 1000 mg/kg b.w./day, it is difficult to state whether the changes were related to the hematological system or the immune system. However, none of these effects need to be considered as adverse

 

In conclusion, The treatment of male and female rats with SPA at the dose levels of 100 and 300 mg/kg bw/day by oral gavage revealed no or no adverse effects. All observed effects of the high dose group were also considered to be incidental or non-adverse, e.g. due to an adaptive response. Based on the results of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of SPA in rats, the no observed adverse effect level (NOAEL) of the test item was determined to be 1000 mg/kg b.w./day for parental, reproduction, and developmental toxicity.

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-(acryloyloxy)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 daysENVIRONMENTAL 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 dose 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

Remarks:

Doses / Concentrations:100, 300, 1000 mg/kg bw/dayBasis:actual ingested

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, non-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)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Effect levels (P0)

1

Key result

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

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

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

Remarks on result:

not determinable due to absence of adverse toxic effects

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, non-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 treatment-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)

Key result

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 422. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats, as an OCED 422 study both covers repeated dose and reproductive (screening) toxicity testing.
In general is should be mentioned ahead that study duration, although not yet subchronic, is prolonged compared to an OECD 407 study (28 days (males) and ca. 43-49 days (females) compared to 28 days). Also, females are pregnant, making them in general more susceptible for toxic effects compared to non-pregnant animals. Hence, testing via an OECD 422 may reveal more distinct effects than testing via an OECD 407, and the observed effects may count as worst case short-term toxicity testing.
As described above, the observed deaths were considered to be either due to a gavage error or incidental, also, no dose-related clinical signs were noted.
The analysis of body weights showed no effects in males, only a decrease in body weights of females (group 3) on day 20 of gestation. 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) (Chou et al., 1998, Minimal risk level (MRLs) for hazardous substances. J. Clean Technol., Environ. Toxicol., & Occup. Med., Vol. 7, No. 1, 1998), body weight loss or decrease in body weight gain of less than 10% is considered non-adverse.
The ophthalmic examinations did not reveal any pathological changes.
A statistically significant decrease in the number of erythrocytes and a statistically significant increase in MCV and MCH levels were observed in males and females in the high dose group compared with the control group. Furthermore, the number of reticulocytes in males from the high dose group was statistically significantly increased. No other hematological parameters showed any significant differences between the treated and the control groups.
Considering various historical control data for hematological parameters for Wistar (CRL:WI) rats (published by Charles River Laboratories) the data indicate that the statistically significant differences observed for MCV, MCH, erythrocytes and reticulocytes in the high dose group are within or similar to the normal biological variation of Wistar rats.In addition, the differences in hematological parameters for the Wistar rats are within or similar to the range of historical control values of CD® rats and F-344 rats (Derelanko, 2008: Derelanko, M.J., 2008, The toxicologist´s pocket handbook. 2nd edition, ISBN 987-142005138-4).Therefore, it can be considered that the differences in hematological parameters in male and female Wistar rats, although statistically significant, are unlikely to be treatment-related effects and thus non-adverse. Also, effects in the erythrocyte/leukocyte system were considered to be non-adverse taking into account information from the satellite groups.
No treatment-related findings resp. findings with no dose-response in biochemical and enzymatic determinations were observed.
No significant changes were observed in the urinalysis parameters except an increase in the number of leukocytes in females from the high dose group. As no dose-response relationship was noted and the effect was not observed in the satellite group, the finding was considered to be non-adverse.
In open field observations, there were no dose-related or non-incidental effects noted. There were no treatment-related effects on responses to visual, tactile, auditory, and pain stimuli. There were no statistically significant differences in the fore- and hindlimb grip strength of males and females in the satellite group, indicating that the observed effect on forelimb strength in high dose males was incidental. No effects were observed for other treated males and females.
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. However, 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.
There were no macroscopic changes in the organs and tissues considered to be dose-related or non-incidental.
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. Therefore, the observed effects were not considered to be treatment-related.

With regard to reproductive performance, all the females and males were mated. 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. There were no significant differences in the fertility parameters between the control and treatment groups.
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. The effects were not considered to be treatment-related. 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. There were no significant differences in the offspring parameters between the control and treatment groups.
The observed clinical signs were not considered to be treatment-related as no dose-response relationship was noted.
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.
There were no treatment-related macroscopic lesions or malformations in the pups found dead and in the pups euthanized on Day 4 post-partum.

In conclusion, the treatment of male and female rats with SPA at the dose levels of 100 and 300 mg/kg bw/day by oral gavage revealed no or no adverse effects. All observed effects of the high dose group were also considered to be incidental or non-adverse, e.g. due to an adaptive response. Based on the results of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of SPA in rats, the no observed adverse effect level (NOAEL) of the test item was determined to be 1000 mg/kg b.w./day for parental, reproduction, and developmental toxicity.

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, “Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.
Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”
There were no significant differences in the fertility parameters between the control and treatment groups, nor in the offspring parameters between the control and treatment groups, nor no treatment-related macroscopic lesions or malformations in the pups found dead and in the pups euthanized on Day 4 post-partum, so no classification as reproductive or developmental toxicant is triggered.

Executive summary:

The objective of this study was to determine and evaluate potential toxicity of potassium-3-sulphonatopropyl acrylate (SPA) following oral administration to rats in a OECD 422 study under GLP. The study involved the determination of the effects of the test item on body weights, food intake, behaviour, clinical, hematological, coagulation, biochemical, enzymatic, urine parameters and the evaluation of gross and histopathological lesions in tissues and internal organs. The study also provided initial information on the effects on reproductive performance of males and females and the development of offspring.

The study was divided into two stages, the dose range-finding study (DRF study) and main study

 

Dose range-finding study (DRF study)

The aim of the DRF study was to select doses for the main study.

It was performed on 20 male and 20 female Wistar rats (Cmdb:WI; outbred).

The animals in the DRF study were divided into four groups. These were:

● one control group (0) which was given distilled water (vehicle control),

● three groups exposed to SPA, i.e.:

· group 1 given SPA at the dose of 100 mg/kg b.w./day

· group 2 given SPA at the dose of 300 mg/kg b.w./day

· group 3 given SPA at the dose of 1000 mg/kg b.w./day

There were 5 males and 5 females in each group.

The test item/ vehicle control was administered every day for 7 days.

 

Course of the DRF study:

Clinical studies: During the experiment, mortality, morbidity, body weights, and food intake were recorded. After 7 days of the experiment, the animals were anesthetized to collect blood for clinical-chemical examinations. Next, they were euthanized and subjected to post-mortem examinations.

Clinical-chemical examinations: After 7 days of the experiment, hematological examinations of peripheral blood, biochemical and enzymatic examinations of serum were conducted.

Post-mortem examinations: All animals used in the DRF study were subjected to these examinations. They involved the observation of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content.

 

Results of the DRF study:

Clinical studies: All animals survived the experiment. No clinical signs were observed. No statistically significant differences in body weights between the treated and the control groups were noticed. Food intake in the treated and the control groups was similar (no statistical analysis).

Clinical-chemical examinations: The hematological, biochemical, and enzymatic examinations did not reveal any statistically significant changes.

Post-mortem examinations: The macroscopic examinations of all animals used in the DRF study did not reveal any lesions.

 

DRF study summary:

Based on the results of the 7-day dose range finding study, the dose levels of 100, 300, and 1000 mg/kg b.w./day were selected for the main study.

 

Main study

The main study was performed on 50 male and 58 female Wistar rats (Cmdb:WI; outbred). The animals used in the main study were divided into four groups. There were 10 males and 12 females in each group. The groups included:

● one control group (0) which was given distilled water (vehicle control),

● group 1 given SPA at the dose of 100 mg/kg b.w./day,

● group 2 given SPA at the dose of 300 mg/kg b.w./day

● group 3 given SPA at the dose of 1000 mg/kg b.w./day

Furthermore, two satellite groups were used. These were:

● one control group (0SAT) which was given distilled water (vehicle control),

● one treated group (3SAT) given SPA at the dose level of 1000 mg/kg b.w./day

The satellite groups were treated for 28 days. After that, they were observed for 14 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects. There were 5 males and 5 females in each satellite group.

After two weeks of initial treatment in the main study, the animals were mated (one male/one or two females). The satellite groups were not mated. The test item/vehicle control was administered to males for 28 days. Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 4 days after delivery (43 – 49 days).

 

Course of the main study:

Clinical studies: During the main study, mortality, morbidity, body weights, and food intake were recorded. Moreover, ophthalmic examinations and behavioural studies were conducted. The latter involved observations of animal behaviour in an open field, responses to stimuli, locomotor activity, and fore- and hindlimb grip strength. 5 adult males and 5 adult females from each group were subjected to the behavioural studies.

The following parameters were determined in offspring: the number of pups in a litter, the numbers of live births and stillbirths, sex of pups, the weight of a litter. Furthermore, abnormalities in body structure and behaviour of pups as well as their mortality up to day 4 of life were recorded. After 28 days (males) or 42 days (SAT groups, males and females) of the experiment, the animals were anesthetized to collect blood for clinical-chemical examinations. Next, they were euthanized and subjected to post-mortem examinations. Pups were euthanized on day 4 after parturition, whereas females on the following day. The females were anesthetized to collect blood for clinical-chemical examinations. After euthanasia, the females and the pups were subjected to post-mortem examinations.

Clinical-chemical examinations: At the end of the experiment, 5 adult males and 5 adult females from each group were subjected to hematological examinations of peripheral blood and bone marrow, coagulation examinations, biochemical and enzymatic examinations of serum, general urinalyses, and examinations of urine sediment.

Post-mortem examinations: The animals used in the main study were subjected to the detailed gross examination which involved observations of the external body surface, all natural apertures and the cranial, thoracic, and abdominal cavities with their content. The numbers of implantation sites and corpora lutea in parental females used in the main study were determined. Absolute and relative weights of the testes, epididymides, and ovaries of all surviving adult animals were determined. Additionally, absolute and relative weights of the brain, thymus, heart, liver, spleen, kidneys, and adrenals of 5 adult males and 5 adult females from each group were determined. The histopathological examination of the following organs was conducted: brain with the cerebellum, spinal cord, muscle with the peripheral nerve, salivary glands, lymph nodes, trachea, thyroid with the parathyroids, stomach, duodenum, jejunum, ileum, cecum, colon, liver, pancreas, spleen, lungs, heart, thymus, kidneys, adrenals, urinary bladder, ovaries, uterus with the cervix, testicles, epididymides, accessory sex glands (prostate with the seminal vesicles and coagulating glands), and all organs showing gross lesions.

Evaluation of the immune system: An evaluation of the immune system of 5 adult males and 5 adult females was based on the results of clinical-chemical examinations and post-mortem examinations of the spleen, thymus, and lymph nodes, as well as the statistical analysis of the absolute and relative weights of the spleen and thymus. The clinical-chemical parameters included blood morphology with a picture of peripheral blood and bone-marrow smears, the concentrations of albumin, total protein, the albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase.

 

Results of the main study:

Clinical studies: During the experiment, mortality of the parental adults was observed. Four animals died during the main study (two males from group 1 and two females from group 3). As for offspring, 6 pups were born dead (2 females in the control group, 3 males in group 2, and 1 male in group 3). The remaining offspring (with the exception of nine pups eaten by mothers) survived the 4-day observation period. The only exception was 1 pup which died one day after the birth (1 female in group 3). These cases of mortality were not test item-related effects.

There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. The animals showed a few pathological clinical signs during the entire experiment. However, these were not test item-related effects. There were no differences in appearance and behaviour of pups between the treated and the control groups.

The analysis of body weights showed a decrease in body weights of females (group 3) on day 20 of gestation and a decrease in body weights of pups in a litter (group 3) on days 0 and 4 of life.

No treatment-related effects were observed during the detailed behavioural studies. On the basis of the detailed clinical observations, made during and after the treatment, and the open field observations, no muscarinic symptoms (weakness, lacrimation, salivation, and anxiety), nicotinic symptoms (diarrhea and motor coordination difficulties), or central symptoms were noticed. Delayed neurotoxicity, i.e. paralysis of the hindlimbs, clumsiness, sensory disturbances, and general paralysis were not observed. The evaluation of responses to stimuli showed no effects of the test item. On the basis of the fore- and hindlimb grip strength measurement, the detailed clinical observations, and the open field observations (body posture, gait, and locomotor activity), it can be concluded that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day (males and females) did not cause any miotoxic changes.

The indices relating to fertility of the parental animals, including mating indices for males and females, fertility indices for males and females, and pregnancy indices in the treated and the control groups were similar, which suggests that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day did not negatively influence fertility of the parental rats.

The test item did not negatively influence the total number of pups in a litter, the numbers of live births and stillbirths in a litter, and the percentages of males and females in a litter in groups 1 and 2.

It proves the lack of harmful effects of the test item at these doses on fetuses. However, the analysis of body weights showed a decrease in body weights of pups in a litter (group 3) on days 0 and 4 of life. The percentages of males and females in a litter in group 3 were similar. The indices relating to survival of offspring of the treated parents show no negative effects on the indices of live births and 4-day survival in comparison with the control group.

On the basis of the results of this study of SPA on rats, it may be concluded that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day (males and females) had no adverse effects, as clinical observations, and effects body weight and food intake measurements may not be considered as adverse.

On the basis of the results of the behavioural studies, it may be concluded that the test item at the doses of 100, 300, and 1000 mg/kg b.w./day (males and females) did not affect the nervous system. Moreover, it did not cause miotoxic changes.

On the basis of pup body weight measurements and the results of the evaluation of reproduction, it may be concluded that the test item at the doses of 100 and 300 mg/kg b.w./day had no adverse effects. A decrease in body weights of pups in litters in group 3 (1000 mg/kg b.w./day) was observed.

Clinical-chemical examinations: Statistical analyses of the clinical-chemical examinations results showed a few statistically significant changes. Some of them were accidental, whereas the others should be perceived as caused by the test item.

Changes which can be perceived as caused by the test item are:

● a decrease in the number of erythrocytes in males and females from group 3,

● an increase in MCV and MCH in males and females from group 3,

● an increase in the number of reticulocytes in males from group 3.

● an increase in the number of polychromatophilic erythroblasts (bone marrow) in males from group 3,

● an increase in the number of orthochromatic erythroblasts (bone marrow) in males from group 3,

● an increase in the total number of cells in the erythrocyte system (bone marrow) in males from

group 3,

● a decrease in the total number of cells in the leukocyte system (bone marrow) in males from group 3,

● a decrease in the leukocyte system/erythrocyte system quantitative ratio (bone marrow) in males

from group 3.

Both coagulation and enzymatic examinations did not reveal any statistically significant changes. As for the biochemical and urine examinations, only accidental changes were stated. The hematological examinations of the satellite groups showed a statistically significant increase in MCV in females from group 3SAT. The increase after the break in the exposure indicated that the test item influence on this parameter was still observed. However, no treatment-related findings resp. findings with a dose-response in biochemical and enzymatic determinations were observed. The remaining clinical-chemical examinations of the satellite group did not reveal any other statistically significant changes.

On the basis of the clinical-chemical examinations, it can be stated that 28-day (males) or 43 – 49-day (females) exposure of rats to potassium-3-sulphonatopropyl acrylate (SPA) at the dose of 1000 mg/kg b.w./day may cause some hematological changes, but none which have to be considered adverse. Increased MCV in females from group 3SAT could be a stable sign. For the doses of 100 and 300 mg/kg b.w./day, no treatment-related changes were observed.

Post-mortem examinations: There were no macroscopic and histopathological changes caused by the test item in examined tissues and organs of the parental males and females from all groups, i.e. 0, 0SAT, 1, 2, 3, and 3 SAT.

On the basis of the results of the study and the statistical analysis of the numbers of corpora lutea and implantation sites in the ovaries of the parental females, it can be concluded that the test item did not cause any changes in the numbers of corpora lutea and implantation sites when compared to the control group. The macroscopic evaluation of the offspring did not reveal any lesions or malformations in live born and stillborn pups.

The analysis of absolute and relative weights of internal organs showed some statistically significant changes however only thymus and spleen absolute and relative weights in males in the high dose group were statistically significantly changed. For females in the high dose group, only spleen absolute and relative weights were statistically significantly increased. Alterations in organ weights are not toxicologically relevant They should not be considered as an adverse effect as no macroscopic abnormalities and no histopathological findings were observed in these organs.

The macroscopic and the histological examinations of the rats treated with potassium-3-sulphonatopropyl acrylate (SPA) at the doses of 100, 300, and 1000 mg/kg b.w./day did not reveal any toxic effects of the test item on reproductive organs and other tissues and organs of the adults. Increased organ weights with no evidence of pathological and histopathological changes should be considered as a non-specific response (i.e. not adverse). There were no macroscopic lesions and malformations in the offspring.

Evaluation of the immune system: The evaluation of the immune system involved a few types of examinations. The first one was the examination of peripheral blood morphology and bone marrow. For this study, some differences in the number of erythrocytes, MCV, MCH, and the number of reticulocytes were stated. A decrease in number of erythrocytes and an increase in MCV and MCH in males and females from group 3, an increase in the number of reticulocytes in males from group 3, and an increase in MCV in females in the satellite groups were noticed. For bone marrow, an increase in the number of polychromatophilic and orthochromatic erythroblasts as well as an increase in the total number of cells in the erythrocyte system in males from group 3 were stated. As for the bone marrow leukocyte system, a decrease in the total number of cells in the leukocyte system in males from group 3 and a decrease in the leukocyte system/erythrocyte system quantitative ratio in males from the group 3 were stated.

The next group of studies relating to the immune system were the assessments of several biochemical parameters such as the concentrations of albumin, total protein, albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of AST, ALT, and alkaline phosphatase. Evaluated as accidental the decreased albumin concentration and the albumin/globulin ratio in males from group 2 were stated. Other biochemical parameters did not change statistically significantly.

On the basis of the clinical-chemical parameters, no impact of the test item at the doses of 100 and 300 mg/kg b.w./day on the immune system was stated. For the dose of 1000 mg/kg b.w./day, it is difficult to state whether the changes were related to the hematological system or the immune system. The macroscopic and the histopathological examinations of the thymus, spleen, and lymph nodes did not reveal any harmful effects of the test item on the immune system. Only thymus and spleen absolute and relative weights in males in the high dose group were statistically significantly changed. For females in the high dose group, only spleen absolute and relative weights were statistically significantly increased. Alterations in organ weights are not toxicologically relevant. They should not be considered as an adverse effect as no macroscopic abnormalities and no histopathological findings were observed in these organs.

On the basis of the clinical-chemical parameters, the histopathological evaluation of the thymus, spleen, and lymph nodes, and the statistical analysis of absolute and relative weights of the thymus and spleen in groups 1 and 2, no impact of the test item on the immune system was stated. For the dose of 1000 mg/kg b.w./day, it is difficult to state whether the changes were related to the hematological system or the immune system. However, none of these effects need to be considered as adverse

 

In conclusion, The treatment of male and female rats with SPA at the dose levels of 100 and 300 mg/kg bw/day by oral gavage revealed no or no adverse effects. All observed effects of the high dose group were also considered to be incidental or non-adverse, e.g. due to an adaptive response. Based on the results of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of SPA in rats, the no observed adverse effect level (NOAEL) of the test item was determined to be 1000 mg/kg b.w./day for parental, reproduction, and developmental toxicity.