Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH guideline 'Detection of Toxicity to Reproduction for Medicinal Products", Chapter 4.1.2

Principles of method if other than guideline:

Pregnant rats (F0-generation) were treated with Olaflur from implantation (6th day of gestation) until weaning (the 21st day of lactation). After spontaneous delivery the F1-generation animals were examined and their development was observed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 27987

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

rat

Strain:

other: CD /Crl: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 54 days
- Weight at study initiation: 191 - 246 g (F0-Females)

- Housing: females were kept singly, the rearing females together with their pups through lactation period up to day 21 of lactation and the mating animals were kept as pairs in MAKROLON cages (type 111
- Diet: Ssniff* R/Z V1324 (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water: tap water was offered ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55% ± 15
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

Route of administration:

oral: gavage

Vehicle:

other: aqua ad iniectabilia

Details on exposure:

- administration volume 10 mL/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each test or control article that was mixed with a carrier, tests by appropriate analytical methods were conducted to determine the concentration and homogeneity of the test or control items in the mixtures. For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at -20 °C or colder until analysis at LPT. At start of the administration period:
Concentration: during treatment with the test item always before administration to the last animal/ dose level group (1 sample/dose level group).
total number of samples: 3

Homogeneity: at start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group).
total number of samples: 9

At termination of the administration period:
Concentration: during the last administration of the test item to the group at a time when the majority of animals was dosed, always before administration to the last animal/dose level group (1 sample/dose level group).
total number of samples: 3

sum of all samples: 15

The samples were labelled with study number, species, type of sample, concentration, sampling time and date.
The method for analysis of fluoride content in aqueous samples was supplied by the Sponsor and is described in LPT Study No, 16180/02 and LPT Study No. 16184/02. The samples were analysed by LPT.
The results of the analyses of the samples showed that the test item-vehicle mixtures were correctly prepared and that concentrations and homogeneity were in good agreement to those expected. The values ranged from 98.0 % to 107.0 % .

Details on mating procedure:

Sexually mature (proved) male rats of the same breed served as partners. The mating ratio was 1:1, male animals were only used for one female partner, female breeding partners were randomly chosen.

F1-generation
For mating the F1-offsprings one male and one female pup (where they were available) were selected and marked at weaning (day 21 of lactation). For each sex, it was the pup which was the median weight for that sex in that litter. The F1-offspring was not mated until they had attained full sexual maturity. Pairing was on a 1 male to 1 female basis, brother-sister pairing was avoided. Each female was transferred to the cage of its appropriate co-group male near the end of the day, where it remained until mating had occurred. Vaginal lavages were taken early each morning, commencing on the day of pairing until mating had occurred and the stage of oestrus observed was recorded in each vaginal lavage. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated as day 0 of gestation. Each female was left with its first designated male for a maximum of 7 nights. If a positive mating signs had not been observed during that time an additional mating period of a maximum of 14 nights was carried out with the same partner. Vaginal lavages were continued to be taken. At the end of the second mating period, if no mating sign had been observed, attempts to breed from the female ceased and the female was returned to an individual cage. Females which showed no positive mating signs at the end of the maximum mating period of 3 weeks of mating were classified in the same way as the pregnant females, i.e. the first day after the mating period was defined as day 0 of gestation.
The time taken to show a positive mating sign and the number of failed opportunities to mate (oestruses passed without any sign of mating) was recorded for each female.

Duration of treatment / exposure:

- 16 days

Frequency of treatment:

- once daily beginning on day 6 of pregnancy until day 21 of lactation

Duration of test:

- 10.03.2004 - 02.09.2004

No. of animals per sex per dose:

- 20 pregnant rats

Control animals:

yes

Details on study design:

In order to obtain the required number of 20 pregnant animals per group, additional animals (in this case '24') were used at the start of the study. All 24 animals were treated and evaluated for pregnancy determination. The reproduction parameters were determined only in 20 animals using the first 20 animals with pregnancy signs.

Maternal examinations:

Clinical Signs:
All the animals were examined at least once daily during the study period until necropsy; during the treatment period the examination was carried out before and after dosing at each time of dosing. The onset, intensity and duration of clinical signs were recorded, particular attention was paid to the first 1 - 2 hours after dosing. Effects were observed until they subsided, if possible.

Viability:
In addition, all the animals were checked for viability early in the morning and again as late as possible in each day. Any animal showing signs of unacceptable debility or distress would have been killed.

Body weight:
F0-generation female body weights were recorded daily during gestation and on days 1, 4, 7, 10, 14, 17 and 21 of lactation to serve as a basis for the calculation of body weight gain during the respective periods.
Post-weaning F1-generation animals were weighed weekly, starting on a suitable day within one week of weaning until termination for males and until mating commenced for females. Mated F1-females were weighed daily during gestation, then on days 1, 4, 7, 10, 14, 17 and 21 of lactation, F1-females that had not shown any positive mating sign were continued on a weekly weighing regime.

Food Consumption:
The quantity of food consumed by each rat was recorded daily for the F0-generation females, from day 0 of gestation (weighed quantity first offered on day 0) until parturition. For females that had delivered, food consumption was recorded weekly over the periods of days 0 - 7 and 7 - 14 of lactation. Relative food consumption was calculated from the absolute food intake and related to the rats' most recently determined body weight. Food consumption was not recorded for F1-generation animals, as this is not required by the ICH guidelines.

Termination:
F0-generation dams
Termination for the F0-generation females was at weaning of their litters (day 22 of lactation).
All F0-generation females which did not deliver were laparotomised on the 24th gestation day and the uteri were examined macroscopically and the implantation sites were determined {according to SALEWSKI). On day 22 of lactation, all dams were sacrificed under ether anaesthesia. They were dissected and inspected macroscopically. The number of implantation sites (according to SALEWSKI) was established per dam. In case of macroscopical findings, the affected organs were preserved for possible histological examination; the corresponding organs of sufficient controls were also preserved for comparison.

Statistics:

The test item-treated groups (groups 2 to 4) were compared statistically with the untreated control group {group 1).
The following Statistical methods were used:
For all numerical values [for example, for body weight of the F0 and F1 parental animals, body weight gain and food consumption (F0-dams), gestation length and the number of implants in F0- and F1-dams, pre-coital time (F1-dams), the number of viable pups throughout the reproduction phase (F1- and F2-generations), entire litter weight and individual pup weight (F1- and F2-generations), data on morphological landmarks
and open-field test (in F1-pups)], homogeneity of variances was tested using the Bartlett chi-square test. When the variances were homogenous, the DUNNETT test {limit of significance: p <= 0.01) was used.
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control
Biometrics, 482-491, September 1964

Indices:

The following parameters were evaluated in F0- and F1-females:
- Number of pregnant females
- Number of females bearing live pups
- Duration of gestation calculated from day 0 of pregnancy
- Number of implantations

Gestation Index [%] = number of dams bearing live pups x I00 /number pregnant

For each litter and group the following indices were determined:
Birth Index [%] = total number of pups born (live + dead) / number of implantation scars x 100
Live Birth Index [%] = number of pups born alive (day 0 or 1 of lactation) / total number of pups born (live + dead) x 100
Lactation Index [%] = number of pups live at day 21 of lactation x 100 / number of pups live on day 4
Viability Index (0-4) [%] = number of pups live on day 4 of lactation / number of pups born alive (day 0 or 1 of lactation) x 100
Overall Survival Index [%] = number of pups alive on day 21 of lactation / total number of pups born (live + dead) x 100

The following parameter was evaluated in F1-females only:
Fertility Index [%] = number of pregnant or siring a litter / number paired x 100

Details on maternal toxic effects:

Maternal toxic effects :yes

Details on maternal toxic effects:
No test item-related mortality was noted.
Behaviour, external appearance and faeces were not influenced by the treatment with 6, 20 or 60 mg Olaflur/kg bw.
Body weight of the dams treated with 60 mg Olaflur/kg bw was slightly reduced by 3 % to 6 % from gestation day 12 until the end of treatment (significant at p < 0.01 reductions were noted on gestation days 20 and 21} when compared with the control animals.
Body weight gain of the high-dosed F0-females was also reduced during gestation from gestation day 7 onwards (range: 9 % to 36 %),
A slight to moderate decrease was noted for the food consumption of the high-dosed female animals (60 mg Olaflur/kg bw) during gestation and lactation (by 6 % to 17 %, statistically significant at p < 0.01 on some gestation days and in lactation week 2).
The treatment did not influence drinking water consumption at 6, 20 or 60 mg Olaflur/kg bw as observed during daily visual appraisal.
Macroscopic inspection revealed no systemic changes connected with the treatment.

Key result

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOEL

Effect level:

> 60 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No test item-related effects were noted for the F1-parents

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOEL

Effect level:

> 60 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No test item-related effects were noted in the F1-parents or F2-generation

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

F0-dams:

Treatment of the F0-dams with 60 mg Olaflur/kg bw from gestation day 6th until the end of lactation did not influence the duration of pregnancy, gestation index, the number of Implantation sites, the average litter size (number of alive and dead pups at birth), the number of viable pups at birth, the birth index, the five birth index, the viability index (0-4 days), the lactation index (4-21 days) and the overall survival index. No influence was noted on the sex ratio of pups (alive and dead) at birth. In addition, no runts and no malformed pups were noted in the dosed groups.

Maternal care: no test item-related influence was noted.

 

Findings for F1-pups:

Body weight:

No test item-related influence was noted for the body weight and the total litter weight of the pups of the dose level groups 6 and 20 mg Olaflur /kg bw The body weight of F1 -pups and the total litter weight of the F1 -pups of the high dose (60 mg Olaflur/kg bw) were slightly reduced, statistically significant (at p <= 0.01) for the total litter weight during the lactation period between days 10 and 21.

However, those variations were not seen in the F2 generation are not considered as a toxicologically relevant effect.

Morphological landmarks:

No test item-related changes were noted.

Functional tests and open-field test:

No test item-related negative influence was noted on the auditory startle reflex, mid-air righting reflex, pupillary reflex, passive avoidance test (learning and memory) and the following parameters of the open-field test: duration of latency and incidence of rearing, sectors entered, defecation boluses at all dose levels.

Final examination of the F1 -weanlings:

No test item-related macroscopic post mortem findings were noted in F1-weanlings, selected for necropsy (2 animals per sex and litter). The external examination of the F1-weanlings at termination did not reveal any lesions.

F1 male and female parents

Mortality:

No mortality was noted.

Clinical signs:

Behaviour, external appearance and faeces of the F1-animals during pre-mating, mating, gestation and lactation were not influenced by the treatment of F0-dams with 6, 20 or 60 mg Olaflur/kg bw/d.

Body weight:

No influence was noted on body weight of male and female animals during the pre-mating and mating periods, and for female animals during the

gestation and lactation periods at any of the tested dose levels.

Pre-coital time:

No test item-related influence was noted.

Macroscopic post mortem findings:

No test item-related macroscopic post mortem findings were noted.

Reproduction

Treatment of the F0 -dams with 6, 20 or 60 mg Olaflur/kg bw/d did not influence the following parameters of the next generation (F1 -animals): the female fertility and gestation indices, the duration of pregnancy, the average litter size, the number of viable pups at birth, birth and live-birth indices. No influence was noted on sex ratio of pups (alive and dead) at birth per group. No differences were noted between the test item groups and the control group with respect to the number of stillbirth, runts and malformed pups. No test item related significantly differences were noted in the viability index (0-4 days), the lactation index (4-21 days) and in the overall survival index between the dosed groups and the control group.

 

F2-pups

Body weight of the F2-pups:

No test item-related changes were noted.

External examination of F2-weanlings:

No test item-related changes were noted. 

Executive summary:

In a developmental toxicity study (LTP, 2005) Olaflur (Aminefluoride 297 -103) was administered to pregnant CD/Crl:CD rats (20 /group, F0 -generation) by oral gavage at dose levels of 0, 6, 20 or 60 mg/kg bw/day from implantation (6th day of gestation) until weaning (the 21st day of lactation). After spontaneous delivery the F1-generation animals were examined and their development was observed employing parameters such as body weight gain, morphological landmarks and functional tests. Dams which did not deliver spontaneously were laparotomised on the 3rd day after the calculated day of delivery. The uterus was examined macroscopically and the implantation sites were determined. One male and one female rat of each litter and group (F1-generation; with a body weight nearest to the mean litter weight) were raised to maturity and mated at the age of 13 weeks. The males were sacrificed after the mating period. After spontaneous delivery, the pups (F2-generation) were weaned and their development was observed employing body weight gain. Dams which did not deliver spontaneously were laparotomised on the 3rd day after the calculated day of delivery. The uterus was examined macroscopically and the implantation sites were determined. The dams and pups were sacrificed after 3 lactation weeks.

The NOEL for maternal (F0-dams) and developmental toxicity (F1-pups) was considered to be 20 mg/kg bw/day and 60 mg/kg bw/d, respectively. At 60 mg/kg bw/day, reduced food intake, reduced body weight and body weight gain were noted in the dams. The variation in the F1-pup development at 60 mg/kg bw/d are not considered to be of toxicological relevance as no such effects were seen in the F2 generation, receiving the substance over a significant longer period of time and over 2 generations.

Since no test item related changes were noted both in F1 male and female parental animals as well as no findings in F2 -pups were discovered, the NOEL for both is considered to be > 60 mg/kg bw/d.

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH guideline 'Detection of Toxicity to Reproduction for Medicinal Products", chapter 4.1.2

Principles of method if other than guideline:

Pregnant rats (F0-generation) were treated with Olaflur from implantation (6th day of gestation) until weaning (the 21st day of lactation). After spontaneous delivery the F1-generation animals were examined and their development was observed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 27987

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: CD /Crl: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
- Age at study initiation: 54 days
- Weight at study initiation: 191 - 246 g (F0-Females)

- Housing: females were kept singly, the rearing females together with their pups through lactation period up to day 21 of lactation and the mating animals were kept as pairs in MAKROLON cages (type 111)
- Diet: Ssniff* R/Z V1324 (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water: tap water was offered ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55% ± 15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: aqua ad iniectabilia

Details on exposure:

- administration volume 10 mL/kg bw/d

Details on mating procedure:

Sexually mature (proved) male rats of the same breed served as partners. The mating ratio was 1:1, male animals were only used for one female partner, female breeding partners were randomly chosen.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each test or control article that was mixed with a carrier, tests by appropriate analytical methods were conducted to determine the concentration and homogeneity of the test or control items in the mixtures. For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at -20 °C or colder until analysis at LPT. At start of the administration period:
Concentration: during treatment with the test item always before administration to the last animal/ dose level group (1 sample/dose level group).
total number of samples: 3

Homogeneity: at start of administration, during (middle) administration and before administration
to the last animal of each dose level group (3 samples/dose level group).
total number of samples: 9

At termination of the administration period:
Concentration: during the last administration of the test item to the group at a time when the majority of animals was dosed, always before administration to the last animal/dose level group (1 sample/dose level group).
total number of samples: 3

sum of all samples: 15

The samples were labelled with study number, species, type of sample, concentration, sampling time and date.
The method for analysis of fluoride content in aqueous samples was supplied by the Sponsor and is described in LPT Study No, 16180/02 and LPT Study No. 16184/02. The samples were analysed by LPT.
The results of the analyses of the samples showed that the test item-vehicle mixtures were correctly prepared and that concentrations and homogeneity were in good agreement to those expected. The values ranged from 98.0 % to 107.0 % .

Duration of treatment / exposure:

- 16 days

Frequency of treatment:

- once daily beginning on day 6 of pregnancy until day 21 of lactation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

6 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

60 mg/kg bw/day (nominal)

Control animals:

yes

Details on study design:

In order to obtain the required number of 20 pregnant animals per group, additional animals (in this case '24') were used at the start of the study. All 24 animals were treated and evaluated for pregnancy determination. The reproduction parameters were determined only in 20 animals, using the first 20 animals with pregnancy signs.

Examinations

Parental animals: Observations and examinations:

Clinical Signs:
All the animals were examined at least once daily during the study period until necropsy; during the treatment period the examination was carried out before and after dosing at each time of dosing. The onset, intensity and duration of clinical signs were recorded, particular attention was paid to the first 1 - 2 hours after dosing. Effects were observed until they subsided, if possible.

Viability:
In addition, all the animals were checked for viability early in the morning and again as late as possible in each day. Any animal showing signs of unacceptable debility or distress would have been killed.

Body weight:
F0-generation female body weights were recorded daily during gestation and on days 1, 4, 7, 10, 14, 17 and 21 of lactation to serve as a basis for the calculation of body weight gain during the respective periods.
Post-weaning F1-generation animals were weighed weekly, starting on a suitable day within one week of weaning until termination for males and until mating commenced for females. Mated F1-females were weighed daily during gestation, then on days 1, 4, 7, 10, 14, 17 and 21 of lactation, F1-females that had not shown any positive mating sign were continued on a weekly weighing regime.

Food Consumption:
The quantity of food consumed by each rat was recorded daily for the F0-generation females, from day 0 of gestation (weighed quantity first offered on day 0) until parturition, for females that had delivered, food consumption was recorded weekly over the periods of days 0 - 7 and 7 - 14 of lactation. Relative food consumption was calculated from the absolute food intake and related to the rats' most recently determined body weight. Food consumption was not recorded for F1-generation animals, as this is not required by the ICH guidelines.

Termination:
F0-generation dams
Termination for the F0-generation females was at weaning of their litters (day 22 of lactation).
All F0-generation females which did not deliver were laparotomised on the 24th gestation day and the uteri were examined macroscopically and the implantation sites were determined (according to SALEWSKI). On day 22 of lactation, all dams were sacrificed under ether anaesthesia. They were dissected and inspected macroscopically. The number of implantation sites (according to SALEWSKI) was established per dam. In case of macroscopical findings, the affected organs were preserved for possible histological examination; the corresponding organs of sufficient controls were also preserved for comparison.

Litter observations:

Assessment of F1 postnatal physical and functional development:
Functional development of the F1 offspring before and/or after weaning was measured by indices of physical maturation such as pinna detachment, eye opening, ear opening, genital development and incisor eruption.
In functional tests [Midair righting reflex, Auditory startie reflex, Pupillary reflex, Open field, Passive avoidance (learning), Passive avoidance (memory; 7 days after learning test) (except open field test] the percentage of not succeeding pups was determined. In addition the pups were examined on morphological landmarks (Pinna detachment, Ear opening, Eye opening, Cleavage of the balanopreputial gland, Vaginal opening , Upper incisor eruption)

Postmortem examinations (offspring):

F1-weanlings not selected for post-weaning assessments:
From each litter, 2 male and 2 female pups (where available) were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 7 % formalin. The carcasses were then discarded.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any pups found that had such an abnormality were necropsied as described. The remaining carcasses were discarded.
F2 -pups:
These animals were killed and discarded after external examination. Any animal with externally visible abnormalities was subjected to gross necropsy, as described above.

Statistics:

The test item-treated groups (groups 2 to 4) were compared statistically with the untreated control group {group 1).
The following Statistical methods were used:
For all numerical values [for example, for body weight of the F0 and F1 parental animals, body weight gain and food consumption (F0-dams), gestation length and the number of implants in F0- and F1-dams, pre-coital time (F1-dams), the number of viable pups throughout the reproduction phase (F1- and F2-generations), entire litter weight and individual pup weight (F1- and F2-generations), data on morphological landmarks
and open-field test (in F1-pups)], homogeneity of variances was tested using the Bartlett chi-square test. When the variances were homogenous, the DUNNETT test {limit of significance: p <= 0.01) was used.
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control
Biometrics, 482-491, September 1964
p = 0.01 & t = 3.03 for 60 to 76 degrees of freedom
In case of heterogeneity of variances, the Student's t-test was carried out, limit of significance was p <= 0.01.
STUDENT'S t-test
p = 0.01 Dt = 2.712 for 38 degrees of freedom
p = 0.01 Dt = 2,716 for 37 degrees of freedom
p = 0.01 D t = 2.724 for 35 degrees of freedom
p = 0.01 D t = 2.733 for 33 degrees of freedom

For the comparison of classification measurements (fertility and gestation indices) the one-tailed FISHER's exact test (p <= 0.05 and p <= 0.01)
and for the sex-ratio index the two-tailed FISHER's exact test (p < 0.05 and p <= O.01) were employed.
For the evaluation of the birth index, live birth index, viability index, lactation index and overall survival index as well as functional tests (except open-field test) chi2-test with Yates' correction for continuity ( p <= 0.05 and p <= 0.01) was employed.
These statistical procedures were used for all data.

Reproductive indices:

The following parameters were evaluated in F0- and F1-females:
- Number of pregnant females
- Number of females bearing live pups
- Duration of gestation calculated from day 0 of pregnancy
- Number of implantations

Gestation Index [%] = number of dams bearing live pups x I00 /number pregnant

For each litter and group the following indices were determined:
Birth Index [%] = total number of pups born (live + dead) / number of implantation scars x 100
Live Birth Index [%] = number of pups born alive (day 0 or 1 of lactation) / total number of pups born (live + dead) x 100
Lactation Index [%] = number of pups live at day 21 of lactation x 100 / number of pups live on day 4
Viability Index (0-4) [%] = number of pups live on day 4 of lactation / number of pups born alive (day 0 or 1 of lactation) x 100
Overall Survival Index [%] = number of pups alive on day 21 of lactation / total number of pups born (live + dead) x 100

The following parameter was evaluated in F1-females only:
Fertility Index [%] = number of pregnant or siring a litter / number paired x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

No test item-related mortality was noted.
Behaviour, external appearance and faeces were not influenced by the treatment with 6, 20 or 60 mg Olaflur/kg bw.
Body weight of the dams treated with 60 mg Olaflur/kg bw was slightly reduced by 3 % to 6 % from gestation day 12 until the end of treatment (significant at p < 0.01 reductions were noted on gestation days 20 and 21} when compared with the control animals.
Body weight gain of the high-dosed F0-females was also reduced during gestation from gestation day 7 onwards (range: 9 % to 36 %),
A slight to moderate decrease was noted for the food consumption of the high-dosed female animals (60 mg Olaflur/kg bw) during gestation and lactation (by 6 % to 17 %, statistically significant at p < 0.01 on some gestation days and in lactation week 2).
The treatment did not influence drinking water consumption at 6, 20 or 60 mg Olaflur/kg bw as observed during daily visual appraisal.
Macroscopic inspection revealed no systemic changes connected with the treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Effect level:

20 mg/kg bw/day

Sex:

female

Basis for effect level:

other: due to reduced body weight of dams treated with 60 mg/kg bw/d

Results: F1 generation

Details on results (F1)

Findings for F1 pups:
Body weight:
No test item-related influence was noted for the body weight and the total litter weight of the pups of the dose level groups 6 and 20 mg Olaflur /kg bw The body weight of F1 -pups and the total litter weight of the F1 -pups of the high dose (60 mg Olaflur/kg bw) were slightly reduced, statistically significant (at p <= 0.01) for the total litter weight during the lactation period between days 10 and 21.
However, those variations were not seen in the F2 generation are not considered as a toxicologically relevant effect.
Morphological landmarks:
No test item-related changes were noted.
Functional tests and open-field test:
No test item-related negative influence was noted on the auditory startle reflex, mid-air righting reflex, pupillary reflex, passive avoidance test (learning and memory) and the following parameters of the open-field test: duration of latency and incidence of rearing, sectors entered, defecation boluses at all dose levels.
Final examination of the F1 -weanlings:
No test item-related macroscopic post mortem findings were noted in F1-weanlings, selected for necropsy (2 animals per sex and litter). The external examination of the F1-weanlings at termination did not reveal any lesions.
F1 male and female parents
Mortality:
No mortality was noted.
Clinical signs:
Behaviour, external appearance and faeces of the F1 -animals during pre-mating, mating, gestation and lactation were not influenced by the treatment of F0 -dams with 6, 20 or 60 mg Olaflur/kg bw/d.
Body weight:
No influence was noted on body weight of male and female animals during the pre-mating and mating periods, and for female animals during the
gestation and lactation periods at any of the tested dose levels.
Pre-coital time:
No test item-related influence was noted.
Macroscopic post mortem findings:
No test item-related macroscopic post mortem findings were noted.
Reproduction
Treatment of the F0 -dams with 6, 20 or 60 mg Olaflur/kg bw/d did not influence the following parameters of the next generation (F1 -animals): the female fertility and gestation indices, the duration
of pregnancy, the average litter size, the number of viable pups at birth, birth and live-birth indices. No influence was noted on sex ratio of pups (alive and dead) at birth per group. No differences were noted between the test item groups and the control group with respect to the number of stillbirth, runts and malformed pups. No lest item related significantly differences were noted in the viability index (0-4 days), the lactation index (4-21 days) and in the overall survival index between the dosed groups and the control group.
 
F2-pups
Body weight of the F2-pups:
No test item-related changes were noted.
External examination of F2-weanlings:
No test item-related changes were noted.
 

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

> 60 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: due to variations on pup weights were not considered to be biological relevant as those were not seen in the F2-generation

2

Key result

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Generation:

F1

Effect level:

> 60 mg/kg bw/day

Sex:

female

Basis for effect level:

other: as no effects in the male and female F1 parents during the pre-mating and mating periods and in F1 dams during gestation and lactation were noted

Results: F2 generation

Effect levels (F2)

Key result

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Generation:

F2

Effect level:

> 60 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: as no effects on the development of the offspring (F2 generation) were noted

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

In a developmental toxicity study (LTP, 2005) Olaflur (Amine fluoride 297 -103) was administered to pregnant CD/Crl:CD rats (20 /group, F0 -generation) by oral gavage at dose levels of 0, 6, 20 or 60 mg/kg bw/day from implantation (6th day of gestation) until weaning (the 21st day of lactation). After spontaneous delivery the F1-generation animals were examined and their development was observed employing parameters such as body weight gain, morphological landmarks and functional tests. Dams which did not deliver spontaneously were laparotomised on the 3rd day after the calculated day of delivery. The uterus was examined macroscopically and the implantation sites were determined. One male and one female rat of each litter and group (F1-generation; with a body weight nearest to the mean litter weight) were raised to maturity and mated at the age of 13 weeks. The males were sacrificed after the mating period. After spontaneous delivery, the pups (F2-generation) were weaned and their development was observed employing body weight gain. Dams which did not deliver spontaneously were laparotomised on the 3rd day after the calculated day of delivery. The uterus was examined macroscopically and the implantation sites were determined. The dams and pups were sacrificed after 3 lactation weeks.

The NOEL for maternal (F0 -dams) and developmental toxicity (F1 -pups) was considered to be 20 mg/kg bw/day and 60 mg/kg bw/d, respectively. At 60 mg/kg bw/day, reduced food intake, reduced body weight and body weight gain were noted in the dams. The variation in the F1-pup development at 60 mg/kg bw/d are not considered to be of toxicological relevance as no such effects were seen in the F2 generation, receiving the substance over a significant longer period of time and over 2 generations.

Since no test item related changes were noted both in F1 male and female parental animals as well as no findings in F2 -pups were discovered the NOEL for both is considered to be > 60 mg/kg bw/d.