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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-03-24 to 2004-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 421
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetic acid, 2-sulfo-, mono-C12-14(even numbered)-alkyl esters, sodium salt
EC Number:
939-512-2
Cas Number:
85681-55-6
IUPAC Name:
Acetic acid, 2-sulfo-, mono-C12-14(even numbered)-alkyl esters, sodium salt
Details on test material:
- Name of test material (as cited in study report): LATHANOL LAL POWDER
- Molecular formula: C14H27NaO5S
- Molecular weight : 330.42
- Substance type: White powder
- Analytical purity: 72.43% and 74.26%
- Lot/batch No.: 7041630 and 228275
- Expiration date of the lot/batch: 2005-02-16 and 2005-05-15
- Stability under test conditions: Formulations stable for 5 hours at room temperature (see analytical details)
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: Not specified.
- Fasting period before study:
- Housing:
Initial - suspended stainless steel cage ( 5/sex/cage)
Mating: one female/one male in suspended stainless seel cage with wire mesh floor
Post-mating - individually in polycarbonate cages with Woody Clean bedding (paper supplied to dams)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 - 25.5°C
- Humidity (%): 30 - 95%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2004-03-29 To: 2004-05-14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial formulations at testing facilities
- Concentration in vehicle: 7.72, 38.7, 194 mg/g
- Amount of vehicle (if gavage): 5 mL/kg
- Purity: Specific gravity = 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy of preparation, homogeneity and stability: formulations prepared weeks 1 and 2
Accuracy and homogeneity only: week 6 and after the in-life phase

Formulation samples dissolved in methanol, ultrasonicated for 10 minutes and diluted to required concentration with methaonl/Milli-Q water.

Analytical conditions:
Column: Nucleosil RP C2 (125 x 4 mm; dp = 0µm)
Gradient:
0 min - 90% Milli-Q water: 10% methanol
10 min - 0% Milli-Q water: 100% methanol
12 min - 90% Milli-Q water: 10% methanol
17 min - 90% Milli-Q water: 10% methanol
Flow: 400 µL/min
Injection volume: 20 µL
Detection: MS
Peak 1: Ionisation source: ESI position 3
Aquisition: MS/MS monitoring the reaction: 307.3 -> 121.0 amu
isolation width: 1.5 amu
Normalised collison energy: 36%
Quantitation on mass: 121 amu
Peak 2: Ionisation source: ESI position 3
Aquisition: MS/MS monitoring the reaction: 335.2 -> 121.0 amu
isolation width: 1.5 amu
Normalised collison energy: 36%
Quantitation on mass: 121 amu

Accuracy:
Week 1 77 - 123%
Week 2 90 - 109%
End of in life phase: 91 - 115%

Homogeneity: Homogeneous - Week 1 Relative standard deviation 4.2 - 11%; Week 2 Relative standard deviation 2.5 - 7.0%

Stability: Formulations at target test material concentration levels of 7.72 and 194 mg/L were found to be stable for at least 5 hours when stored at room temperature (relative difference between 0.5% and 9.0%).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one to one
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
Males: 39 days (2 weeks pre mating, during mating and up to the day before necropsy)
Females: 42 - 46 days (2 weeks prior to mating, during mating, during post coitum and at least 3 days of lactation)
Frequency of treatment:
Daily, 7 days a week
Duration of test:
Maximum 46 days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on 28-day range finder and 90-day repeat oral dose study. the high dose level of 1000 mg/kg bw/day was selected as no severe effects were expected based on the results of the repeat dose studies. In addition 1000 mg/kg bw/day is the maximum dose level that can be tested under the Guideline. 200 mg/kg bw/day was chosen as the mid-dose level as only minor toxicological effects were expected. The low dose of 40 mg/kg bw/day was chosen yp obtain a linear distribution.
- Rationale for animal assignment): random based on weight

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on first day of exposure and weekly thereafter. mated females on days 0, 7, 14 and 21 of gestation, and during lactation on days 1 and 4

FOOD CONSUMPTION: Yes
- Time schedule: Weekly. During the mating period analysis of food consumption was suspended. Food consumption of mated females was measured on gestation days 0, 7, 14 and 21 and during lactation on days 1 and 4.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 4 post partum or shortly thereafter. Males after mating or minimum 28 days completed.
- Organs examined: cervix, clitoral gland, coagulation gland, epidymides, ovaries, pituitary gland, preputial gland, prostate gland seminal vesicles, stomach, testes, uterus, vagina, all gross lesions.
Females: number of implantation sites and corpora lutea
Organ weights in males: epididymides, testes
Histopathology: stomach, ovaries, testes, epididymides, all gross lesions

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations:No
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
No statistical analysis was performed on the histopathology findings.
Indices:
Percentage mating
No. females mated/No. females paired x 100
Fertility Index
No. females pregnant/No. females paired x 100
Conception rate
No. pregnant females/No. females mated x 100
Gestation Index
No. females bearing live pups/No. pregnant females x 100
Duration of gestation
No. days between confirmation of mating and the beginning of parturition
Percentage live males at first litter check
No. live males pups at first litter check/No. live pups at first litter check x 100
Percentage live females at first litter check
No. live females pups at first litter check/No. live pups at first litter check x 100
Percentage of postnatal loss days 0-4 post partum
No. dead pups on day 4 post partum/ No. live pups at first litter check x 100
Percentage of breeding loss day 5 until weaning
No. dead pups between days 5 and 21 post partum/No. live pups on day 4 post partum x 100
Percentage live males at weaning
No. live male pups on day 21 post partum/No. live pups on day 21 post partum x 100
Percentage live females at weaning
No. live female pups on day 21 post partum/No. live pups on day 21 post partum x 100
Viability Index
No. pups surviving on day 4 post partum/ No. pups born alive x 100
Weaning Index
No. live pups on day 21 post partum/No. live pups on day 4 post partum x 100




Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality/Viability:
One female at the high dose level was killed in extremis. At necropsy, this female shwed two foetal resoprtions and haemorrhagic blood in the left uterus horn. No other unscheduled deaths occurred during the study period.
Clinical signs:
At 1000 mg/kg bw/day all males and females showed salivation during almost the complete study period. rats were observed incidentally for a few animals treated at 1000 mg/kg bw/day.
One male of the control group showed lethhargy, hunched posture, piloerection, and curled tail apex at the end of the study period. As this male was not exposed to the test substance, these findings were considered to be incidental.
Incidental findings that were noted included, alopecia of several body parts, scabs on several body parts, and diarrhoea. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
Body weights:
Body weight gain was statistically significantly decreased for males on day 8 of treatment at 1000 mg/kg bw/day. This was probably caused by the lower food intake during the first week of pre-mating.
Bodyweights and bodyweight gain of treated males up to 200 mg/kg bw/day and females up to 1000 mg/kg bw/day remained in the same range as controls over the study period.
Food consumption
Food consumption (absolute and/or relative) of the highest dose group was statistically significantly decreased for males during the first week of treatment and for females during the first two weeks of treatment and days 7-14 of post-coitum.
An increase in absolute food consumption was seen during the first two weeks of treatment for males treated at 40 mg/kg bw/day and during the second week of treatment for males treated at 1000 mg/kg bw/day. The increase at 1000 mg/kg bw/day was probably a response to recover from the decreased food consumption during the forst week. These increases were very slight and were thus considered to be of no toxicological relevance.

Pathology
Macroscopic evaluation
Forestomach abnormalities were observed for all males and two females of the 1000 mg/kg bw/day dose grou. These consisted of thickened forestomach surface, and isolated reddish foci on the forestomach.
The female that was killed in extremis showed two foetal resorptions and haemorrhagic/clotted blood in the left uterus horn.
Incidental findings included pelvic dilation of the kidneys, testes and epididymides redced in size, preputial glands reduced in size, enlarged mandibular lymph node, preputial glands enlarged and showing an irregular surface, dark red soft nodule on liver, watery-clear cysts on the ovaries, uterus containing fluid and diaphragmatic hernia of the liver. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment related distribution they wee considered changes of no toxicological significance.
Organ weights
Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.
Microscopic examination
Hypeplasia of the squamous epithelium of the forestomach was seen at a minimal to moderate degree in all animals of the high dose group. This alteration was accompanied by minimal to moderate degree of lymphogranulocytic inflammation in the forestomach in four males and two females in the high dose group. In two males of the high dose group there were also erosions - minimal or slight, of the forestomach mucosa. These findings were the historical correlates to the macroscopic observations found at necropsy in the stomach.
Assessment of testes for spermatogenesis stage revealed no deficiencies in the seminiferous epithelium of high dose rats.
In the non-pregnant female (low-dose group) and the male suspected of infertility (low-dose group) there were no findings inthe reprductive organs which could have contributed to suspected infertility.
The remainder of the microscopic findings recorded were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Breeding data
Breeding parameters were unaffected by treatment up to 1000 mg/kg bw/day
The number of live and dead pups at first litter check (day 1 of lactation), postnatal loss between days 0 and 4 post partum and viability index were similar for control and treated groups.
The increased postnatal loss and decreased viability index at 40 mg/kg bw/day was mainly due to the loss of one complete litter and was thus considered not to be toxicologically relevant.

Pup development
Pup development was unaffected by treatment up to 1000 mg/kg bw/day. mean body weights were similar for control and treated groups.
Incidental findings consisted of a small, cold or pale appearence, little or no milk, absent tail, blue or red discolouration of several body parts, dying and cannibalism. Macroscopic examination of the pups revealed no milk, cannibalism, tail missing, and a small appearence. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at high dose
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1 Reproduction data

Number of females

Group 1

Group 2

Group 3

Group 4

 

Control

40 mg/kg bw/day

200 mg/kg bw/day

1000 mg/kg bw/day

Paired

10

10

10

10

Mated

10

10

10

10

Non-pregnant

0

1

0

0

Pregnant

10

9

10

10

All foetal resorptions

-

-

-

1

Number of litters on Day 1

10

9

10

9

 

Table 2 Fertility

 

Group 1

Group 2

Group 3

Group 4

 

Control

40 mg/kg bw/day

200 mg/kg bw/day

1000 mg/kg bw/day

Percentage mating

No. females mated/No. females paired x 100

100.00

100.00

100.00

100.00

Fertility Index

No. females pregnant/No. females paired x 100

100.00

90.00

100.00

100.00

Conception rate

No. pregnant females/No. females mated x 100

100.00

90.00

100.00

100.00

Gestation Index

No. females bearing live pups/No. pregnant females x 100

100.00

100.00

100.00

90.00

Applicant's summary and conclusion

Conclusions:
Gavage treatment of male and female Wistar rats with the test material at dose concentrations of 40, 200 and 1000 mg/kg bw/day revealed parental toxicity at the high dose level. Reproduction and pup development were not affected up to and including 1000 mg/kg bw/day.
Based on the results the NOAEL for parental toxicity was 200 mg/kg bw/day and the NOAEL for reproductive effects is 1000 mg/kg bw/day.
Executive summary:

In an OECD Guideline 421 Screening test for reproductive and developmental toxicity four treatment groups of ten Wistar rats/sex/group were administered the test article at dose levels of 40, 200, or 1000 mg/kg/day. One additional group of ten animals/sex served as the control and received the vehicle, propylene glycol only. The vehicle or test article was administered to all groups once daily via oral gavage. The males were dosed for 39 days, beginning 14 days prior to pairing. Dosing of the females began 14 days prior to pairing, through the mating period, up to and including lactation Day 4.

Observations of the parental animals included clinical signs, body weights and body weight change, and food consumption during the premating/mating, gestation, and lactation periods, and parturition and litter data. Observations of the offspring included survival at birth and during lactation, individual pup body weights, and gross abnormalities. At study termination, necropsy examinations were performed on all parental animals, and organs and tissues were collected, weighed and examined for select groups. On Day 4 post partum pups were examined externally, euthanized, and discarded.

The analytical evaluation of the formulation samples confirmed that they were homogenous and at the targeted concentration required.

Parental toxicity was observed at the high dose level. The treatment related findings observed were increased incidence of clinical signs (salivation and rales), decreased bodyweight gain and food consumption, macroscopic abnormalities of the forestomach) consisting of thickened forestomach, irregular forestomach surface, and isolated reddish foci on the forestomach) and microscopic abnormalities of the forestomach (consisting of hyperplasia of the squamous epithelium, sometimes accompanied by lymphogranulocytic inflammation or erosions of the forestomach mucosa). There were no treatment-related findings at the low and mid dose levels. The NOAEL for maternal toxicity is 200 mg/kg bw/day

Reproductive, fertility, parturition, and litter data, including external pup observations did not reveal any changes that could be considered treatment related.

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for reproductive, and developmental toxicity was considered to be 1000 mg/kg/day, the highest level tested and the limit dose designated by the guideline.

 In accordance with CLP EC Regulation No. 1272/2008, the test material is not classified for reproductive toxicity.