Alkylbenzenesulphonic acid compound with alkanolamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

For effects on reproduction, studies on the acid are available. No effects on reproduction were reported in the OECD 422 study (MOE 2008) at the 400 mg/kg bw (highest dose). This is similar to the findings in two three -generation studies on the sodium salt of the acid, where the parental and foetal NOAEL values were 170 mg/kg and 350 mg/kg, the highest doses tested.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: the tested substance is the acid part of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

1) Test animals- Supplier: Orient Bio Co. Ltd. 143-1, Sangdaewon-dong, Jungwon-ku, Sungnam, Gyunggi-do, 462-120 Korea- Age at study initiation: 7-week-old animals for male and female
- No. of animals at receipt: 57 for male and female
- Body weights at study initiation: 212.5–243.8 g for males and 147.6 -168.6 g for females
- Age at the first day of treatment: 8 weeks for male and female
- Body weight range at the first day of treatment: 274.2∼311.1 g for males and 175.7∼213.4 g for females
- All animals were visually examined on acquisition. Only the animals remained in good physical condition during the 6-day acclimatization in the animal room were selected for the test.
2) Environmental condition
- Temperature 23 +/- 3 deg C, relative humidity of 50 +/- 10%; ventilation of 10 to 20 times/hours; light/dark cycle 12 h/12 h
- All animals used in this study were cared for in accordance with the principles outlined in the "Guide for the Care and Use of Laboratory Animals", a NIH publication.
3) Monitoring- Room temperature was generally in the range 20-26 deg C, relative humidity was generally in the range 40-60%. No significant deviations, which can affect the experiment, were observed.
4) Housing and identification of animals- Equal or less than five for the quarantine and acclimatization
- Equal or less than two for the pre-mating, treatment and recovery period5) Diet, water and bedding material
- Pelleted maintenance diet and tap water ad libitum; no contaminants (analysed)

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on mating procedure:

- Premating exposure period for males and females: 2 weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test article of the highest dose group was mixed with water for injection, and the low dose group's test article was prepared by dilution of that of the highest dose group. The test article solutions were prepared once a day before completion of the analytical method validation, and after completion the test article was formulated over once a week.

Duration of treatment / exposure:

From 2 weeks before mating to the end of -the mating period for male (at least 28 days)From 2 weeks before mating to day 4 of lactation including the mating and gestation periods for female- Post exposure period: 15 days in both sexes

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
nominal conc. 0, 100, 200, and 400 mg/kg bw/day (Dosing volume 10mL/kg/day)
Basis:
nominal conc.

No. of animals per sex per dose:

10 males and females for 100 and 200 mg/kg bw/day and 16 males and females for 400 mg/kg bw/day (10 was for test group and 6 was for recovery group), 16 males and females for vehicle control (10 was for test group and 6 was for recovery group)

Control animals:

yes

Details on study design:

Treatment- Dose levels determined in a pilot toxicity study of dodecylbenzenesulfonic acid in rats- Constant dosage volume of 10 mL/kg bw/day: calculated with Path/Tox system according to the basis of recently measured body weight.- Dosing of both sexes was begun at 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were dosed after the mating period at least until the minimum total dosing period of 28 days had been completed. Daily dosing of the parental females was continued throughout pregnancy and at least up to day 4 post-partum.

Parental animals: Observations and examinations:

- Mating: The day verified by sperm in a vaginal rinse was designated as day 0 of pregnancy. Based on the results, following indices were calculated. Mating index = (No. of animals with successful copulation / No. of mated animals) × 100 Fertility index = (No. of impregnating animals / No. of animals with successful copulation) × 100 Pregnancy index = (No. of pregnant animals / No. of animals with successful copulation) × 100
- Observation on gestation and parturition: Abortion, premature delivery and dystocia or prolonged parturition were observed.
- Observation on parturition date: Gestation length, delivery index, litter size, sex ratio, external anomalies of live pups were observed.
- Observation during the lactation period: Nursing behaviours of dams and viability of pups were observed. Following data were obtained from these observations. Pregnancy periodDelivery index: No. of dams with live newborns/ No. of pregnant damsx100Newborn survival index:Newborn mortality index:Viability index: No of live pups on day 4 of lactation/ No of neonates at birthx100Body weight in all survival animals on 0 and 4 days afterbirth.

Postmortem examinations (parental animals):

- Gross findings: At scheduled termination, all live animals were anaesthetized by isoflurane inhalation, blood samples taken and then terminated by exsanguinating the abdominal aorta. Complete gross post mortem examinations were performed on all animals.
- Organ weights: Absolute organ weights were measured and their relative organ weights (organ-to-body weight ratios) were calculated from the terminal body weight for the following organs of selected six animals when they were sacrificed. (Brain, pituitary gland, adrenal gland,spleen, kidneys, heart, thymus, lungs and thyroid (with parathyroid)). However, the following organs were weighed in all animals except the non-pregnant.(liver, salivary gland, testis, epididymis, seminal vesicle, prostate, ovary, uterus).
- Histopathological examination: Histopathological examination was performed on the following tissues from animals in the vehicle control and 64 mg/kg bw/day groups. abnormal lesion, skin (included mammary gland in females), urinary bladder, pancreas, mesenteric lymph node, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, adrenal glands,mandibular lymph node, thyroid (included parathyroid), aorta, thymus, heart, lungs, tongue, trachea, esophagus, sciatic nerve, skeletal muscle, sternum, femur, eye with optic nerve, haderian gland, brain, pituitary gland, spinal cord (thoracic) and nasal cavity. Additional examination was conducted in spleen, femur and sternum of the 4 and 16 mg/kg bw/day groups, since treatment-related findings were observed in these organs. Neutral buffered 10% formalin was used for fixation and preservation, except testis, epididymides and eyeballs. Bouin’s fixative was used for testis and epididymides and Davidson’s solution for eye ball. Lungs and urinary bladder were inflated with fixative prior to immersion in fixative. And then organs were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically.

Statistics:

- Body weights, food consumption, organ weights, and clinical pathology : means the standard deviation of each mean. - Bartlett's test : analyzing for homogeneity of variance- Dunnett's t test : analyzing for the significance of inter-group differences- Analysis of Variance : analyzing for homogeneous data- Kruskal-Wallis test : analyzing for Heterogeneous data- Dunn's Rank Sum test : analyzing for the significance of inter-group differences between the control and treated groups- F test : analyzing the data of recovery groups for homogeneity of variance- Dunnett's t test : analyzing for homogeneous data- Dunn's Rank Sum test : analyzing for the significance of inter-group differences- t test : analyzing for Heterogeneous data- Kruskal-Wallis test : analyzing for the significance of inter-group differences between the control and treated group-Statistical analyses were performed by comparing the different dose groups with the vehicle control group using Path/Tox System.- p<0.05 or p<0.01

Histopathological findings: non-neoplastic:

no effects observed

Reproductive performance:

no effects observed

There were no treatment-related changes in precoital time, mating index, fertility index and pregnancy index.Results for F0- Precoital time, fertility and mating data: There were no statistically significant differences compared with the controls.- Gross findings: There were no chemical- related findings in all treatment groups.- Histopathological findings: There were no chemical- related findings in all treatment groups.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects reported

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects reported

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects reported

Key result

Reproductive effects observed:

no

Conclusions:

There were no treatment-related changes were observed in copulation, fertility and pregnancy indices, gestation length, the number of corpora lutea and implantation, delivery index. Based on these effects, the NOAEL (no-observed-adverse-effect levels) for fertility was 400 mg/kg bw/day.

Executive summary:

Effects on Fertility

In reproductive toxicity study performed according to the reproduction/developmental toxicity screening test [OECD TG 422] conditions and dose were same asrepeated dose toxicity. A distilled water for injection was used as vehicle.

No statistically significant differences were seen in the following parameters examined: gestation length, the number of corpora lutea and implantation, delivery index, precoital time, fertility, mating data and in the histopathological examination. The NOAEL was 400 mg/kg bw/day in both sexes.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Oral administration of the amine at doses upto 1000 mg/kg bw during gestation did not lead to effects on development. In addition no maternal toxicity was observed (Dow 2004). For the acid a study according to OECD 422 showed no effects on the foetuses at the highest concentration tested, 400 mg/kg bw , via the oral route (MOE 2008). No effects on development were seen in a study with the acid where the substance was applied dermally from day 0 to 20 of gestation (1980). The maternal NOAEL in this study was 20 mg/kg bw based on inhibition of body weight gain related to local skin effects. No developmental effects in the pups were observed at the highest dose tested, 400 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

other: the tested substance is the acid part of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD TG 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in rats"

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

1) Test animals- Supplier: Orient Bio Co. Ltd. 143-1, Sangdaewon-dong, Jungwon-ku, Sungnam, Gyunggi-do, 462-120 Korea- Age at study initiation: 7-week-old animals for male and female- No. of animals at receipt: 57 for male and female- Body weights at study initiation: 212.5-243.8 g for males and 147.6 -168.6 g for females- Age at the first day of treatment: 8 weeks for male and female- Body weight range at the first day of treatment: 274.2-311.1 g for males and 175.7-213.4 g for females- All animals were visually examined on acquisition. Only the animals remained in good physical condition during the 6-day acclimatization in the animal room were selected for the test.
2) Environmental condition- Temperature 23 +/- 3 deg C, relative humidity of 50 +/- 10%; ventilation of 10 to 20 times/hours; light/dark cycle 12 h/12 h- All animals used in this study were cared for in accordance with the principles outlined in the "Guide for the Care and Use of Laboratory Animals", a NIH publication.
3) Monitoring- Room temperature was generally in the range 20-26 deg C, relative humidity was generally in the range 40-60%. No significant deviations, which can affect the experiment, were observed.
4) Housing and identification of animals- Equal or less than five for the quarantine and acclimatization- Equal or less than two for the pre-mating, treatment and recovery period5) Diet, water and bedding material- Pelleted maintenance diet and tap water ad libitum; no contaminants (analysed)

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test article of the highest dose group was mixed with water for injection, and the low dose group's test article was prepared by dilution of that of the highest dose group. The test article solutions were prepared once a day before completion of the analytical method validation, and after completion the test article was formulated over once a week.

Details on mating procedure:

Premating exposure period for males and females: 2 weeks

Duration of treatment / exposure:

From 2 weeks before mating to the end of -the mating period for male (at least 28 days)From 2 weeks before mating to day 4 of lactation including the mating and gestation periods for female- Post exposure period: 15 days in both sexes.

Frequency of treatment:

daily

No. of animals per sex per dose:

10 males and females for 100 and 200 mg/kg bw/day and 16 males and females for 400 mg/kg bw/day (10 was for test group and 6 was for recovery group), 16 males and females for vehicle control (10 was for test group and 6 was for recovery group)

Control animals:

yes

Details on study design:

Treatment- Dose levels determined in a pilot toxicity study of dodecylbenzenesulfonic acid in rats.- Constant dosage volume of 10 mL/kg bw/day: calculated with Path/Tox system according to the basis of recently measured body weight.- Dosing of both sexes was begun at 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were dosed after the mating period at least until the minimum total dosing period of 28 days had been completed. Daily dosing of the parental females was continued throughout pregnancy and at least up to day 4 post-partum.

Maternal examinations:

-Observation of pregnancy and delivery-Pregnancy period-No. of implantation and corpus lutea-Delivery index=(No. of dams with live newborns/ No. of pregnant dams) x 100

Fetal examinations:

-No. of perinatal death-No. of live young on day 0 and 4 at postpartum-No. of pups with gross lesions-No. of pups with runts-Viability index at day 4 of postpartum=(No. of live pups at day 4/ No. of live pups at birth) x 100-Body weights of pups on day 0 and 4 postpartum

Statistics:

- Body weights, food consumption, organ weights, and clinical pathology : means the standard deviation of each mean. - Bartlett's test : analyzing for homogeneity of variance- Dunnett's t test : analyzing for the significance of inter-group differences- Analysis of Variance : analyzing for homogeneous data- Kruskal-Wallis test : analyzing for Heterogeneous data- Dunn's Rank Sum test : analyzing for the significance of inter-group differences between the control and treated groups- F test : analyzing the data of recovery groups for homogeneity of variance- Dunnett's t test : analyzing for homogeneous data- Dunn's Rank Sum test : analyzing for the significance of inter-group differences- t test : analyzing for Heterogeneous data- Kruskal-Wallis test : analyzing for the significance of inter-group differences between the control and treated group-Statistical analyses were performed by comparing the different dose groups with the vehicle control group using Path/Tox System. - p<0.05 or p<0.01

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Results for F0 and F1No statistically significant differences were seen in the following parameters examined: gestation length, the number of implantation, delivery index, the number of live and dead pups, live pups/implantation ratio, dead pups/implantation ratio, pre-implantation loss, post-implantation loss, sex ratio, viability index, number of neonates with external anomalies, and body weights of pups on post-natal day 0 and day 4. On the other hand, a statistically significant decrease in corpora lutea was observed in the 400 mg/kg bw/day group. But this change was in normal range and unrelated to dodecylbenzenesulfonic acid dosing.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Results for F0 and F1No statistically significant differences were seen in the following parameters examined: gestation length, the number of implantation, delivery index, the number of live and dead pups, live pups/implantation ratio, dead pups/implantation ratio, pre-implantation loss, post-implantation loss, sex ratio, viability index, number of neonates with external anomalies, and body weights of pups on post-natal day 0 and day 4. On the other hand, a statistically significant decrease in corpora lutea was observed in the 400 mg/kg bw/day group. But this change was in normal range and unrelated to dodecylbenzenesulfonic acid dosing.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects reported

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

There were no treatment-related changes in all parameters of offsprings during the parturition and lactation periods. Based on these effects, the NOAEL (no-observed-adverse-effect levels) for developmental toxicity was 400 mg/kg bw/day of F1 pups.

Executive summary:

Developmental Toxicity

In reproductive toxicity study performed according to the reproduction/developmental toxicity screening test [OECD TG 422] conditions and dose were same as repeated dose toxicity. The offspring delivered by chemical-treated rats had been observed until day 4 of postpartum. There were no statistically significant differences were seen in the following parameters: the number of live and dead pups, live pups/implantation ratio, dead pups/implantation ratio, pre-implantation loss, post-implantation loss, sex ratio, viability index, number of neonates with external anomalies, and body weights of pups on post-natal day 0 and day 4. There were no treatment-related changes in all parameters of offsprings during the parturition and lactation periods and the NOAEL for developmental toxicity was 400mg/kg bw/day for F1 pubs.

Reference 2

Endpoint:

developmental toxicity

Type of information:

other: the tested substance is the amine part of the compound

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sexually mature adult, 10-11 weeks of age and weighing approximately 200-250 grams.Each animal was evaluated by a laboratory veterinarian or a trained animal/toxicology technician, under the direct supervision of a lab veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were placed in their cages (one per cage) and allowed to acclimate to the laboratory conditions for approximately four days prior to the start of dosing. The animal rooms of the facility are designed to maintain adequate environmental conditions (temperature, humidity, and photocycle).Animals were housed, one per cage, in stainless-steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Room temperature was recorded daily. The relative humidity was maintained within a range of 47.5-50.7%. The room temperature was maintained at 22 ± 1°C (with a maximum permissible excursion range of ± 3°C). These values were within the laboratory recommended range for rats. A 12-hour light/dark photocycle was maintained in all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour. Cages had wire-mesh floors and were suspended above catch pans. Cages contained feed containers and pressure activated, nipple-type watering systems.Animals were stratified based upon gestation day 0 body weights and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean body weights and standard deviations at the start of dosing. Animals placed on study were uniquely identified via subcutaneously-implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers. If a transponder stopped functioning or was lost, it was replaced with a new transponder that correlated with the unique animal number.Animals were provided LabDiet® Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. The results of the feed and water analysis indicated that there were no contaminants present at levels that would interfere with the conduct of the study or interpretation of the results.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose solutions were prepared in deionized water at concentrations of 25, 75 and 250 mg/ml and administered at a dose volume of 4 ml/kg body weight in order to achieve the targeted dose levels. Dose volumes were adjusted daily based on individual body weights. Dose solutions were prepared periodically based on stability data.The homogeneity of DIPA in vehicle was not conducted since the test material was in solution.Diisopropanolamine was found to be stable in water at concentrations ranging from 0.607 to 17.0 mg/ml for at least 25 days.Analysis of all dosing suspensions from the second mix were done using high performance liquid chromatography (HPLC) with RI and external standards to determine concentrations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of all dosing solutions from the first mix revealed mean concentrations of DIPA ranging from 105% to 97.2% of the targeted concentrations.

Details on mating procedure:

Sexually mature, adult virgin females were naturally mated with males of the same strain at the supplier’s facility. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males’ cages. The day on which a vaginal plug was detected was considered day 0 of gestation. Day 0 body weights were provided by the supplier, and maintained in the study record. Rats arrived in our laboratory on gestation day (gd) 1 or 2.

Duration of treatment / exposure:

Groups of approximately 25 time-mated female CD rats were administered DIPA by gavage at dose levels of 0, 100, 300, and 1000 mg/kg/day on days 6-20 of gestation.

Frequency of treatment:

daily

Duration of test:

days 6-20 of gestation

No. of animals per sex per dose:

25 female rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Clinical examinations were conducted daily throughout the study period. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), unusual swelling or masses and animal behavior. In addition, at least, once each day a cage-side examination was conducted and to the extent possible, the following were evaluated: skin, fur, mucousmembranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water.Body weights were recorded on GD 0 (by the supplier), and daily during the dosing period, and at necropsy (GD 21). Statistical analysis of body weights was performed using data collected on GD 0, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.Feed consumption was recorded for all animals for GD 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21 by weighing feed containers at the start and end of a measurement cycle.Anatomic PathologyNecropsyOn day 21 of gestation, all surviving females (not fasted) were euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies were counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification. The sex of all fetuses were recorded and the body weight of all viable fetuses determined. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual administration of a sodium pentobarbital solution. At least one-half of all the fetuses in each litter were chosen randomly using a computer program, and a visceral for visceral examination was conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). Remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol, and double stained with Alcian Blue and Alizarin Red S for cartilage and bone respectively, according to methods based on Trueman et al. (1999). After staining, skeletons were macerated and cleared. Thorough evaluations of the fetal skeletons were conducted on theremaining fetuses not selected for visceral examination. However, a fetus may have been intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it was deemed that such examination could provide more meaningful data about a suspected abnormality. All fetal alterations were classified as variations or malformations. A variation was defined as a divergence beyond the normal range of structural constitution that may not have adversely affected survival or health. A malformation was defined as a permanent structural change that may have adversely affected survival, development or function and/or which occurred at a relatively low incidence in the specific species/strain. Maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment.

Maternal examinations:

The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities opened and the viscera examined. The stomach, liver, and kidneys were dissected from the carcass and incised. Any obvious gross pathologic alterations were recorded, and the weights of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues.

Ovaries and uterine content:

A detailed examination of the reproductive tract were performed and the number and position of implantations, viable fetuses, dead fetuses, and resorptions were recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicated a very recent death as evidenced by a lack external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted. The uteri of females lacking visible implantationswere stained with a 10% aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Fetal examinations:

The sex of all fetuses were recorded and the body weight of all viable fetuses determined. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual administration of a sodium pentobarbital solution. At least one-half of all the fetuses in each litter were chosen randomly using a computer program, and a visceral for visceral examination was conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). Remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol, and double stained with Alcian Blue and Alizarin Red S for cartilage and bone respectively, according to methods based on Trueman et al. (1999). After staining, skeletons were macerated and cleared. Thorough evaluations of the fetal skeletons were conducted on the remaining fetuses not selected for visceral examination. However, a fetus may have been intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it was deemed that such examination could provide more meaningful data about a suspected abnormality.All fetal alterations were classified as variations or malformations. A variation was defined as a divergence beyond the normal range of structural constitution that may not have adversely affected survival or health. A malformation was defined as a permanent structural change that may have adversely affected survival, development or function and/or which occurred at a relatively low incidence in the specific species/strain. Maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment.

Statistics:

Maternal body weights, maternal body weight gains, organ weights (absolute and relative), fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analyses of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction(Miller, 1966) were performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched. Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction. The number of corpora lutea, implantations, and litter size were evaluated using a nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios were analyzed using a binomial distribution test. Females lacking visible implantations at the scheduled necropsy were excluded from the appropriate analyses. Potential outliers were statistially identified using a sequential method (alpha = 0.02; Grubbs, 1969), but were notexcluded unless justified by sound scientific reason unrelated to treatment. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha level. Final interpretation of the data considered statistical analyses and whether the results were consistent with other biological and pathological findingsand historical control values

Indices:

Calculation of Pre- and Post-Implantation Loss• Pre-implantation loss* = ((No. corpora lutea – implantations)/No. corpora lutea) x 100• Post-implantation loss* = ((No. implantations – viable fetuses)/No. implantations) x 100

Historical control data:

Available for review with study results

Details on maternal toxic effects:

Maternal toxic effects:no effectsDetails on maternal toxic effects:Examinations performed on all animals revealed a small number of incidental findings, none of which were considered treatment-related findings. Differences in body weight and body weight gain between treated groups and controls were not statistically significant. Differences in food consumption between treated groups and controls were not statistically significant. Differences in terminal body weight, liver, or kidney weights between treated groups and controls were not statistically significant. All observations were considered to be spontaneous alterations, unassociated with exposure to diisopropanolamine.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:There were no statistically significant treatment-related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal body weights, or gravid uterine weights at any dose level.There was a significant shift in sex ratio in the control group when analyzed by binomial distribution.There were no statistically significant differences in the incidence of any fetal alteration in any of the treated groups compared to controls. The small number of alterations observed in fetuses from dams administered DIPA either occurred at low frequencies and/or were not dose related. Malformations observed among fetuses from the control group included an extra thoracic centra, vertebrae, and rib (in one fetus), a filamentous tail (in another fetus from the same litter), and anasarca (in two pups from a second litter). Additionally, a third litter had a fetus with anasarca, a misshapen heart, a bilateral ventricular septal defect, and an ectopic left kidney. In treated litters, one fetus had a left hydroureter (low-dose group), one fetus had three class II wavy ribs (9th, 10th, and 11th thoracic rib; mid-dose group), and one fetus had a bilateral hydroureter (high-dose group). These malformations were not considered to be treatment-related because of their low incidence and lack of dose response.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity highest dose tested

Key result

Abnormalities:

no effects observed

Developmental effects observed:

not specified

There were no treatment-related in-life observations, and body weights and food consumption were not statistically different than control animals.
There were likewise no findings relative to organ weight or pathology, and reproductive parameters were unremarkable except a significant shift in sex ratio in the control group when analyzed by binomial distribution. Fetal alterations were not statistically different than the control group. There were sporadic alterations across dose groups, although a lack of dose-response and low incidence led to the conclusion that the alterations were not treatment-related.

Conclusions:

There was no maternal or developmental toxicity noted at dose levels up to 1000 mg/kg/day. The NOEL, therefore, was 1000 mg/kg/day, the highest dose level tested.

Executive summary:

Groups of 25 time-mated female CD rats were administered solutions of the amine part by gavage at targeted dose levels of 0, 100, 300, or 1000 mg/kg/day on gestation day (GD) 6 through 20 in order to evaluate the maternal and developmental toxicity potential of this compound. In-life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Maternal liver, kidney and gravid uterine weight were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed, and examined for external alterations. Approximately one half of the fetuses were examined for visceral and cranofacial alterations while skeletal examinations were conducted on the remaining fetuses.

Oral gavage administration of DIPA at dose levels up to and including 1000 mg/kg/day produced no treatment-related maternal toxicity and no indication of developmental toxicity at any dose level. Therefore, under the conditions of this study, the no-observedeffect level (NOEL) for maternal toxicity and developmental toxicity was 1000 mg/kg/day, the highest dose level tested.

Reference 3

Endpoint:

developmental toxicity

Type of information:

other: the tested substance is the acid part of the compound

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: LAS was applied to depilated areas on the chests and backs of female rats 12- 18 weeks of age. Five to six hours prior to treatment an exposure site (roughly 24 cm2) in the dorsothoracic region of each animal from group II through IX was clipped to a length of 1 mm. The animals were reclipped every 48 hr throughout the study. Group I animals were unclipped, group II animals were clipped but not treated and group III animals were clipped and treated with tap water. The mated female rats were treated daily from day 0 through day 20 of gestation. A 0.5-ml sample of the appropriate concentration of LAS and/or tap water was applied once daily to the clipped area and spread with a gloved finger over as much of the exposure site as
possible. Each application was carried out slowly over a 3-min period. In the 1, 5 and 20% LAS groups (groups VII, VIII and IX, respectively corresponding to 20, 100 and 400 mg/kg/day) the test material was allowed to remain on the backs of the animals for 30 min. after which it was removed with warm tap water. The test material was not removed from the backs of the animals in the 0.05, 0.1 and 0.5% LAS groups (groups IV, V and VI corresponding to 1, 2 and 10 mg/kg/day). Animal body weight and food consumption were determined during the treatment period. Daily observations were also made for toxicological effects.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

- Sex: female- Age: 12-18 weeks old

Route of administration:

dermal

Details on exposure:

LAS was applied to depilated areas on the chests and backs of female rats 12- 18 weeks of age. Five to six hours prior to treatment an exposure site (roughly 24 cm2) in the dorsothoracic region of each animal from group II through IX was clipped to a length of 1 mm. The animals were reclipped every 48 hr throughout the study. Group I animals were unclipped, group II animals were clipped but not treated and group III animals were clipped and treated with tap water.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

The mated female rats were treated daily from day 0 through day 20 of gestation. A 0.5-ml sample of the appropriate concentration of LAS and/or tap water was applied once daily to the clipped area and spread with a gloved finger over as much of the exposure site as possible. Each application was carried out slowly over a 3-min period.

Duration of treatment / exposure:

0-20 gestation meet

Frequency of treatment:

Daily

Duration of test:

sacrifice at day 21 of gestation

Control animals:

yes, concurrent no treatment

Details on study design:

Other: LAS was applied daily during the gestation period.Test group was devided into six groups by test material concentration.0.05, 0.1 and 0.5% (1, 2 and 10 mg/kg/day) (group Ⅳ Ⅴ Ⅵ) active ingredient were applied and allowed to remain on the skin. 1, 5 and 20%(20, 100 and 400 mg/kg/day) (group Ⅶ Ⅷ Ⅸ) active ingredient were applied and removed after a 30-min exposure period

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Pregnancy rates: 100% except group Ⅳ(95%) Ⅸ(95.2%).Mortality: No mortality was observed.Mean body weight: in group Ⅸ was slightly reduced from days 12-21 of the gestation period Skin change: slight skin discoloration was observed from day 3 to 6 of gestation (group Ⅶ), slight erythema and dry skin was observed from days 3 to 6 of gestation (group Ⅷ).

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean number of corpora lutea, implantations, viable foetuses or resorptions : not found in any of the treatment groups. Viability and size: no relationship to LAS administration was evident.Number of thoracic and lumbar vertebrae and phalanges: no significant differences between the LAS-treated group and control group.Soft tissue: distended renal pelvis, distended ureters, ectopic testis, and distended bladder were observed but these symptoms were not considered to be related to LAS application.Skeletal and visceral abnormalities: not found in any of the treatment groups.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In maternal, there were only slight change of body weight and skin change. In foetals, no significant differences between the LAS – related group and control group were observed. Thus, LAS was not induced teratogenic or embryotoxic effects.

Executive summary:

NOAEL Maternal: 1% (20 mg/kg bw d)

NOAEL teratogenicity: 20% (400 mg/kg bw d)

Results:

Maternal toxicity:

The dams treated with 20% and 5% showed inhibition of body weight gain and local skin effects.

Teratogenicity:

There were no indications of teratogenic or embryotoxic effects at any level in either group tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Mode of Action Analysis / Human Relevance Framework

The data on the acid show that no effects on reproduction, fertility and development occur at the highest dose of 1000 mg/kg bw. The NOAEL for these effects will therefore in a worst case approach be based on the NOAEL of the acid and is set at 400 mg/kg bw. No effects on development were seen in absence of parental toxicity.

Justification for classification or non-classification

Based on the available information, there is no need to classify the substance for effects on reproduction and/or development