N,N-bis(carboxymethyl)-L-glutamic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

August 2008 - May 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported study (see also section 13 for additional comment)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar Crl:WI(Han) (outbred, SPF-Quality), Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 5-6 weeks
- Weight at study initiation: mean weight of male groups 168-171 g; those of females 149-152 g
- Fasting period before study: not applicable
- Housing:
* Pre-mating: animals were housed in groups of 4 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
* Mating: females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
* Post-mating: males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 4 animals/cage. Females were
individually housed in Macrolon cages (MIII type, height 18 cm).
* Lactation Offspring was kept with the dam until termination.
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7 - 24.0
- Humidity (%): 22-88; cleaning procedures in the room might have caused the temporary fluctuations above the optimal level of 70% for relative humidity.
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 2008 To: May 2009

Route of administration:

oral: feed

Vehicle:

water

Details on exposure:

DIET PREPARATION
The test substance was dissolved in water (Elix, Millipore S.A.S., Molsheim, France). If the amount of test substance was "x" gram in the high dose
groups (Groups 4 and 5), then the volume of water to be added was approximately 4 times "x" gram. For up to 15 kg diet, the test substance was
dissolved in water at a maximum of 25 wt%. For amounts higher than 15 kg and up to 20 kg diet, the test substance was dissolved in water at a
maximum of 33 wt%. The same amount of vehicle was used for preparation of the formulations of all groups. The obtained formulation was mixed with some powder feed (premix). Subsequently, this premix was mixed with the bulk of the diet. Elix water (approximately 15% in total, corrected
for the amount of vehicle used for preparation of the formulation) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.
Stability of test substance in the diet GLDA included to standard rodent diet at dose levels of 1500, 5000 and 15000 ppm was stable for 23 days at room temperature (determined on diets prepared before start of treatment). GLDA-Na4 included to diet supplemented with 1000 ppm zinc-
carbonate at a dose level of 15000 ppm was stable for 22 days at room temperature. Diets were prepared once a week. Diets were kept at room
temperature in the diet store room in the animal house.

Diet groups 1-4: Standard powder rodent diet (SM RIM-Z from SSNIFF@ Spezialdiaten GmbH, Soest, Germany; total content of zinc: 93.5 zinc per kg
diet from 26 August until 23 October 2008, and 88.6 g zinc per kg diet from 24 October until 30 December 2008).
Group 5: Powder rodent diet (SM RIM-Z from SSNIFF@ Spezialdiaten GmbH, Soest, Germany) supplemented with 1000 ppm zinc-carbonate (total
content of zinc: 605 mg zinc per kg diet from 26 Augustus 2008 until 23 October 2008 and 609 mg zinc per kg diet from 24 October 2008 until 30 December 2008)

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: max of 15 days
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no info
- Verification of same strain and source of both sexes: no info
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 post coitum
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the diet were conducted before the start of the study, and in diets prepared in week 1, 11, 22 and 33 (full 2-generation study).
Samples of diet preparations were analysed to check homogeneity and accuracy of preparation. Stability under storage conditions (room
temperature) was also determined. Pellets were ground using a grinding device. A subsample of approximately 5 g was taken from the ground
samples and accurately weighed into a vessel. After addition of 50 ml water, the samples were shaken at 225 rpm for 60 minutes. The extracts
were filtered through a 0.45 pm filter (Spartan 3010.45 RC, Whatman, Dassel, Germany) and diluted with copper(l1)-acetate solution by a factor of 2, 3 or 4. This dilution was stabilized for at least 15 minutes and additionally filtered through a 0.45 pm filter (Spartan 3010.45 RC,
Whatman). If necessary, the filtered solutions were further diluted with water to obtain concentrations within the calibration range.
The extracts were diluted with copper(l1)-acetate to form a copper complex with GLDA which was analysed using high performance liquid
chromatography with UV detection. Separation was performed on a Symmetry C18 column using a mixture of acetic acid, copper(l1)-acetate and DMAC in water-acetonitrile as mobile phase.

Duration of treatment / exposure:

From 70 days before mating (F0 generation) until Day 21 of lactation of pups of the second generation.

Frequency of treatment:

Continuously (diet)

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters (10 week-exposure)
- Selection of parents from F1 generation when pups were 21 days of age
- Age at mating of the mated animals in the study: ca. 13 weeks (3 weeks weaning and 10 weeks exposure)

Remarks:

Doses / Concentrations:
0, 1500, 5000 and 15,000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

24
In the first generation, 5 groups were used: 0, 1500, 5000, 15,000 and 15,000 ppm plus additional Zn
In the second generation, 4 groups were used: 0, 1500, 5000, and 15,000 ppm (without additional Zn)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on a RF study with levels of 0, 1000, 3000 and 10,000 ppm
- Rationale for animal assignment (if not random): computer randomization
- Other:

Positive control:

Not needed

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; during gestation on days 0, 4, 7, 11, 14, 17 and 20, during lactation on days 1, 4, 7, 14 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly (except during breeding) and during gestation on days 0, 4, 7, 11, 14, 17 and 20, during lactation on days 1, 4, 7, 14 and 20
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No (subjective appraissal only)

OTHER: Reproduction processes. Male number paired with, mating date, confirmation of pregnancy, and delivery date were recorded.
Heamatology and clinical chemistry in blood samples collected at necropsy (fasted rats); urinalysis in urine samples collected at necropsy (fasted
rats).

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 21 days prior to initiation of the mating period and until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also performed to determine the stage of estrous. An analysis of the female cycle pattern was
reported.

Sperm parameters (parental animals):

Sperm samples were taken from the proximal part of the vas deferens (right):
1. Sperm motility was assessed from all samples using the TOX Ivos sperm analyzer.
2. Sperm smears were fixed from all samples for morphological evaluation. Abnormal forms of sperm from a differential count of at least 200
spermatozoa (if possible) per animal were recorded. Evaluation was performed for ten randomly selected males of the control group and high dose
group(s).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); part of the excess pups were killed on day 4 for skeletal examination, the other excess pups were killed on day 21 post partum and discarded.

GROSS EXAMINATION OF DEAD PUPS: Yes (see further)
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Mortality / Viability: The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If
possible, defects or cause of death were evaluated.
Clinical signs: At least once daily detailed clinical observations were made in all animals.
Body weights: Live pups were weighed during lactation on Days 1, 4, 7 and weekly thereafter.
Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).
Balanopreputial separation: Each selected male pup (24 rats/group) was observed for balanopreputial separation (prepuce opening) beginning on
postnatal Day 35 (Korenbrot, 1977). Examination of the males was continued daily until balanopreputial separation was present. The body weight of
each male was recorded on the day of acquisition of balanopreputial separation.
Vaginal perforation: Each selected female pup (24 rats/group) was observed for vaginal perforation (vaginal opening) beginning on postnatal Day 25
(Adams, 1985). Examination of the females was continued daily until vaginal perforation was present. The body weight of each female was recorded on the day of acquisition of vaginal perforation.
Anogenital distance: In the absence of treatment related effects in the sex ratio or sexual maturation of the F1-generation, no measurement of the
anogenital distance was performed in the F2- pups.

Postmortem examinations (parental animals):

One testis and one epididymis (left) from all males were removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left
testis and epididymis were weighed, homogenized and evaluated for sperm numbers using the TOX Ivos sperm analyzer. The sperm production rate was calculated using the method described by Blazak et al. (1985). All samples of the control group and high dose group(s) were evaluated.

For all paired females, the number of implantation sites in the uterus was assessed.

After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special
attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues
and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands) and examined: Kidneys, Prostate gland, Seminal vesicles, Cervix, Coagulation gland, Testes, Epididymides, Uterus, Vagina, Ovaries, and all gross lesions.

The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Adrenal
glands, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Kidneys, Testes, Liver, Thyroid, Ovaries, Uterus, Pituitary gland.

Postmortem examinations (offspring):

Stillborn pups and pups dying between birth and Day 4 of lactation were sexed and dissected (including the heart and the brain examined by a mid-
coronal slice) by a technique described by Stuckhardt and Poppe (1984). If a skeletal anomaly was suspected, the pups were eviscerated, skinned,
cleared and stained with Alizarin Red S as described by Dawson (1926) and examined. These examinations were only performed when practically
possible (e.g. in the absence of cannibalism, autolysis). For pups found dead or killed in extremis on Day 5 of lactation to weaning, a gross necropsy
was performed (including sex determination) and gross lesions were saved for possible future histopathological examination, if applicable.
Culled offspring was sexed and externally examined with emphasis on developmental morphology. The stomach was examined for the presence of
milk. All non-selected F1- and F2-weanlings were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were
recorded. All gross lesions were collected and placed in 10% buffered formalin.

The following organ weights (and terminal body weight) were recorded from one randomly selected pup/sex/litter (both generations) on the
scheduled day of necropsy, if possible: Brain, Spleen, Thymus. After weighing, samples of these organs were fixed in 10% buffered formalin for
possible future analysis.

For the F1-pups, the following slides were examined by a pathologist: - the spleen from six female F1-pups/litter of control Group 1 and high
dose Groups 4 and 5 and the thymus from six F1-pups/sex/litter of control Group 1 and high dose Groups 4 and 5.
For the F2-pups, the following slides were examined by a pathologist: - spleen and thymus from one randomly selected F2-pup/sex/litter of
control Group 1 and high dose Group 4.

Per litter, if possible, one male and one female of the F1-offspring selected for culling (approximately Day 4 of lactation) were chosen at random
and processed for detection of possible skeletal variations or malformations (excluding ossification). The carcasses were eviscerated, skinned and fixed in labeled containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons. Each
eviscerated and skinned pup, following fixation in alcohol, was macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with
Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (1926). The skeletal examination was made
following this procedure. The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Merck, Darmstadt, Germany) as a preservative. Only F1-offspring of Groups 1, 4 and 5 was examined. Since no treatment-related effects were noted, skeletal examination was not extended to F1-offspring of Groups 2 and 3.

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as lowest level of statistical significance.
The T-test was applied for sperm concentrations in the testis and epididymis. The percentage of motile spermatozoa, progressive motile spermatozoa and sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test (p<0.05), the Wilcoxon test was applied to
the data to compare the treated groups to the control group.

Reproductive indices:

Percentage mating males = (Number of males mated x 100) / (Number of males paired)
Percentage mating females = (Number of females mated x 100) / (Number of females paired)
Fertility index males = (Number of males generating a pregnancy x 100) / (Number of males paired)
Fertility index females = (Number of pregnant females x 100) / (Number of females paired)
Conception rate = (Number of pregnant females x 100) / (Number of females mated)
Gestation index = (Number of females bearing live pups x 100) / (Number of pregnant females)

Offspring viability indices:

Percentage live males = (Number of live male pups at First Litter Check x 100) / (Number of live pups at First Litter Check)
Percentage live females = (Number of live female pups at First Litter Check x 100) / (Number of live pups at First Litter Check)
Percentage of postnatal loss Days 0-4 = (Number of dead pups on Day 4 post-partum x 100) / (Number of live pups at First Litter Check)
Percentage of breeding loss Da5 until weaning = (Number of dead pups between Days 5 and 21 post-partum x 100) / (Number of live pups on Day 4 post-partum)
Percentage live males at weaning = (Number of live male pups on Day 21 post-partum x 100) / (Number of live pups on Day 21 post-partum)
Percentage live females at weaning = (Number of live female pups on Day 21 post-partum x 100) / (Number of live pups on Day 21 post-partum)
Viability index = (Number of live pups on Day 4 post-partum x 100) / (Number of pups born alive)
Weaning index = (Number of live pups on Day 21 post-partum x 100) / (Number of live pups on Day 4 post-partum)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): no treatment-related effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): no treatment-related effects

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): no treatment-related effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no treatment-related effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no treatment-related effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no treatment-related effects

ORGAN WEIGHTS (PARENTAL ANIMALS): In the F0 females treated at 15000 ppm with or without zinc added (Groups 4 and 5) statistically
significantly higher mean kidney weights were observed (absolute and relative to body weight). In males, mean kidney weight (relative to body
weight) was statistically significantly higher in animals of Group 5 (with zinc added). In the F1-animals, at 15000 ppm, mean kidney weight relative to body weight was statistically significantly increased in males and females. Mean absolute kidney weight was also significantly increased
in females of this group.

GROSS PATHOLOGY (PARENTAL ANIMALS): no treatment-related effects

HISTOPATHOLOGY (PARENTAL ANIMALS): In the kidneys of the F0-animals there was a slight increase in the incidence of minor grades - minimal
or slight, of corticomedullary tubular basophilia in Group 5 (15000 ppm with zinc) males which was however not statistically significant. In the F1-generation corticomedullary tubular basophilia at minor degrees of severity was increased in the kidneys of males of all treated groups, only
reaching statistical significance in Group 4 (15000 ppm; p < 0.01). There was also an increase in renal cortical tubular dilation at minor degrees of severity in females of Group 4 (p < 0.01).

OTHER FINDINGS (PARENTAL ANIMALS): no treatment-related effects in haematology, clinical chemistry and urinalysis

Dose descriptor:

NOAEL

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased kidney weight and minimal to slight histopathological renal changes

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING): no treatment-related effects

CLINICAL SIGNS (OFFSPRING): no treatment-related effects

BODY WEIGHT (OFFSPRING): no treatment-related effects

SEXUAL MATURATION (OFFSPRING): no treatment-related effects

ORGAN WEIGHTS (OFFSPRING): In F1-pups, statistically significantly lower mean absolute and relative spleen and thymus weights were noted in
female pups following treatment at 15000 ppm (with or without added zinc). In male pups of these groups, a statistically significant lower mean
relative thymus weight was observed.In the F2-pups, at 15000 ppm, mean spleen and thymus weight (absolute and relative to body weight) were
decreased in male and female pups (not always statistically significant).

GROSS PATHOLOGY (OFFSPRING): no treatment-related effects

HISTOPATHOLOGY (OFFSPRING): All histopathologic findings recorded in the spleen and thymus of the F1- and F2-pups were considered
to be within the normal range of background pathology encountered in Wistar Han rat pups of this age and strain.

OTHER FINDINGS (OFFSPRING): no

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 15 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction or developmental effects

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 15 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction or developmenatl effects

Reproductive effects observed:

not specified

When corrected for mean test article intake the NOAEL of 5000 ppm corresponds to 287 -389 mg and 380-894 mg GLDA per kg body weight per day for males and females, respectively. The NOAEL of 15000 ppm corresponds to 908-1229 and 1230-2822 mg GLDA per kg body weight per day for males and females, respectively.

Conclusions:

Treatment of male and female Wistar Han rats at dietary dose levels of 1500, 5000 and 15000 ppm GLDA-Na4 revealed parental toxicity at 15000
ppm based on increased mean relative kidney weight in F0- and F1-adults and minimal to slight microscopic renal changes in F1-adults.
Reproduction, skeletal morphology (up to and including Day 4 post-partum) and development were unaffected up to 15000 ppm. As there were no
differences in the groups at 15000 ppm with and without added zinc, it was concluded that the addition of zinc was not necessary to compensate for possible reproductive toxicity of GLDA-Na4, if any, due to its chelating (zinc-binding) properties.

Executive summary:

A two-generation reproduction toxicity study of GLDA-Na4 was conducted in rats by dietary administration. The dose levels were based on a 14-day range finding study in which rats were exposed to 0, 1000, 3000 and 10000 ppm by dietary administration. Based on the results of the dose range finding study, dose levels for the main study were selected to be 0, 1500, 5000 and 15000 ppm GLDA-Na4.

F0 -generation: After acclimatization, five groups of 24 male and 24 female Wistar Han rats were exposed via dietary administration to the test substance at dose levels of 1500 (Group 2), 5000 (Group 3) and 15000 ppm (Groups 4 and 5). Rats from the control group (Group 1) received preparations of standard rodent SM R/M-Z diet in pelleted form without GLDA. Groups 2-4 were exposed to GLDA-Na4 included into standard pelleted rodent diet (total content of zinc: 88.6-93.5 mg per kg diet). Group 5 animals received pelleted diet preparations with the test substance at the high dose level included in standard rodent diet supplemented with 1000 ppm zinc-carbonate (total concentration of zinc: 605-609 mg per kg diet). Group 5 with additional dietary zinc was added to the study to compensate for possible (repro-) toxic effects, if any, due to the chelating properties of GLDA. The amount of zinc to be added was calculated based on a study by Swenerton & Hurley (1971). F0-males and F0-females were exposed to the test substance from 10 weeks prior to mating and exposure was continued until euthanasia (males) or one day before euthanasia (females). F0 -females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation, litters were reduced in size to eight pups (4 per sex when possible) by random culling of F1 -pups. After weaning, one F1-male and one F1-female of each litter of Groups 1 -4 were selected for mating with a pup of another litter of the same dose group to produce a F2-generation.

F1-generation: Four groups of 24 male and 24 female Wistar Han rats were exposed via dietary administration to the test substance at dose levels of 1500 (Group 2), 5000 (Group 3) and 15000 ppm (Group 4). GLDA-Na4 was included into standard pelleted rodent diet (total content of zinc: 83.8 mg per kg diet). No additional Zn-supplemented group was added to this phase of the study. Rats from the control group (Group 1) received preparations of standard rodent SM R/M-Z diet in pelleted form without GLDA-Na4. The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation, and directly when they started eating solid food and following weaning. After weaning, animals were treated for a minimum 70 days prior to mating and continuing until euthanasia (males) or one day before euthanasia (females). F1-females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation, litters were reduced in size to eight pups by random culling of F2-pups. The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.

The following parameters were evaluated: mortality, clinical signs, body weights, food consumption, clinical laboratory investigations (including collection of blood samples for possible future zinc analysis; females only), reproduction processes, observations on offspring, macroscopy, skeletal examination of offspring, organ weights, and histopathology. Chemical analyses of diets were conducted five times to assess accuracy and homogeneity and twice to assess stability (over 22 and 23 days at room temperature). Accuracy, homogeneity and stability of the diets were demonstrated by the results of the diet analyses.

Results:

at 1500 ppm (Group 2): Parental, reproduction and developmental toxicity: No findings.

at 5000 ppm (Group 3): Parental, reproduction and developmental toxicity: No findings.

at 15000 ppm without or with zinc (Groups 4 and 5, respectively): Parental toxicity: increased mean kidney weight was observed in F0- and F1- adults and slight histopathological renal changes were noted for F1 -adults. The histopathological changes were of minor degrees of severity and consisted of an increase in cortical tubular dilation in females and an increase of corticomedullary tubular basophilia in males. Reproduction and developmental toxicity: No findings.

It was concluded that treatment of male and female Wistar Han rats at dose levels of 1500, 5000 and 15000 ppm GLDA-Na4 revealed parental toxicity at 15000 ppm based on increased relative kidney weight in F0- and F1-adults and minimal to slight microscopic renal changes in F1-adults treated with 15000 ppm. Reproduction, skeletal morphology (up to and including Day 4 post-partum) and development were unaffected up to 15000 ppm. As there were no differences in the groups at 15000 ppm with and without additional zinc, it was concluded that the addition of zinc was not

necessary to compensate for possible reproductive toxicity of GLDA-Na4, if any, due to its chelating (zinc-binding) properties. Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established as being 5000 ppm. The reproduction and developmental NOAELs were established as being at least 15000 ppm. When corrected for mean test article intake the NOAEL of 5000 ppm corresponds to 287-

389 mg and 380-894 mg GLDA-Na4 per kg body weight per day for males and females, respectively. The NOAEL of 15000 ppm corresponds to 908 -1229 and 1230 -2822 mg GLDA-Na4 per kg body weight per day for males and females, respectively.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

908 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

One well performed and reported study available.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Treatment of male and female Wistar Han rats at dietary dose levels of 1500, 5000 and 15000 ppm GLDA-Na4 revealed parental toxicity at 15000

ppm based on increased mean relative kidney weight in F0- and F1-adults and minimal to slight microscopic renal changes in F1-adults.

Reproduction, skeletal morphology (up to and including Day 4 post-partum) and development were unaffected up to 15000 ppm. The parental No Observed Adverse Effect Level (NOAEL) was established as being 5000 ppm. The reproduction and developmental NOAELs were established as being at least 15000 ppm. When corrected for mean test article intake the NOAEL of 5000 ppm corresponds to 287 -389 mg and 380-894 mg GLDA-Na4 per kg body weight per day for males and females, respectively. The NOAEL of 15000 ppm corresponds to 908 -1229 and 1230 -2822 mg GLDA-Na4 per kg body weight per day for males and females, respectively.

Short description of key information:
In a 2-generation dietary study in rats no reproduction or developmental effects were observed at a dietary level up to 15,000 ppm GLDA-Na4. This corresponds to 908 -1229 and 1230 -2822 mg GLDA-Na4 per kg body weight per day for males and females, respectively.

Justification for selection of Effect on fertility via oral route:
Well performed and reported study with the read across substance GLDA-Na4.

Effects on developmental toxicity

Description of key information

In both rats and rabbits, there was no sign of (prenatal) developmental toxicity at levels up to 300 mg/kg bw (rabbit - gavage) or up to 15,000 ppm 
(rat - diet). The latter level corresponded to 1230 -2668 mg GLDA-Na4 per kg bw for females.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

May-July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study; this is a repeat study of the study carried out in 2008 (Beekhuizen, 2009) at the request of the French authorities (see letter of French authorities attached in section 13).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test System: female albino rabbits, New Zealand White (NZW) strain (SPFQuality), from a non-inbred laboratory colony. Nulliparous, non-pregnant
and untreated females were used at initiation of the study. Stock male NZW rabbits were used for mating with the females. These males were
adult and proven fertile. After mating these males were placed back in their stock and might be used for future studies.
Rationale: this species and strain of rabbit has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Source: Charles River, Chatillon sur Chalaronne, France.
Number of animals: F0-generation: 88 females, F1-generation: 697 fetuses.
Age at delivery: females were approximately 18 weeks.
Acclimatization: at least 5 days prior to pairing.
Conditions: environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were recorded in the raw data; these variations were
considered to have had no effect on the outcome of the study.
Accommodation: females were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Shelters were removed for females that were excessively soiling their shelters.
Diet: free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy). In addition, pressed hay (BMI,
Helmond, the Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study
period.
Water: free access to tap-water.
Diet and water evaluation for contamination and/or nutrients was performed according to facility standard procedures. There were no contaminant levels found that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method: oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.
Frequency: once daily for 7 days per week, approximately the same time each day with a maximum of 4 and a half hours difference between the earliest and latest dose on these days.
Exposure period: from Day 7 to Day 28 post-coitum, inclusive.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (25 May 2012), according to a validated method (Project 483244) using an HPLC-UV method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all
concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

From 8.00 am onwards, one female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating (which usually occurred within 5 minutes). This day was designated Day 0 post-coitum. Twenty-four male rabbits were used for mating with the 88 female rabbits.

Duration of treatment / exposure:

From Day 7 to Day 28 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 4 and a half hours difference between the earliest and
latest dose on these days.

Duration of test:

Animals surviving to scheduled necropsy were euthanised on Day 29 post-coitum.

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were based on a prenatal developmental toxicity study in NZW rabbits (Project 487520) in which doses of 30, 100 and 300 mg/kg body weight/day were tested. No fetal malformations or developmental variations were noted in this study. As such the developmental NOAEL for GLDA was established to be at least 300 mg/ kg bw/day. An increased number of non-pregnant females was, however, noted in all treated groups compared to the control group but these numbers were within the normal range or just below the historical control data and still considered normal. A dose response relationship was not present. Based on the reduced number of pregnancies the French Authorities, however, concluded that this study was of too low quality and should be repeated with at least one dose level lower than 30 mg/kg bw/day. In consultation with the sponsor, dose levels of 20, 75 and 300 mg/kg were selected.

Maternal examinations:

Mortality / Viability: at least twice daily.
Clinical signs: at least once daily from Day 0 post-coitum onwards. The time of onset, degree and duration was recorded. All symptoms were graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present; Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,
grade 3 = severe, grade 4 = very severe.
Cage debris was examined to detect abortion or premature birth
Body weights: Days 0, 4, 7, 10, 13, 16, 20, 23, 26, 29 post-coitum.
Food consumption: Days 0-4, 4-7, 7-10, 10-13, 13-16, 16-20, 20-23, 23-26 and 26-29 postcoitum.
Water consumption: subjective appraisal was maintained during the study until 11 June 2012, and a quantitative assessment was introduced from 12 June 2012 onwards based on a possible treatment related effect.
External, thoracic and abdominal examinations were performed for the detection of macroscopic abnormalities. All abnormalities were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Animals found dead or sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
External:
Each viable fetus was examined in detail, sexed and weighed. All live fetuses were euthanized by subcutaneous injection of 0.1 mL pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) in the area between the scapulas. Nonviable fetuses (the degree of autolysis was
minimal or absent) were examined, crown-rump length measured and weighed. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible).
Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. The sex of all fetuses was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin. The heads from the remaining one-half of the fetuses in each litter were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.
Skeletal:
The eviscerated fetuses from Groups 1 and 4, following fixation in alcohol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. All specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

Statistics:

The following statistical methods were used to analyze the data:
- 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.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

Indices:

For each litter the following calculations were performed:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) x 100
number of corpora lutea

Post-implantation loss (%) = (number of implantation sites - number of live fetuses) x 100
number of implantation sites

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was noted at 300 mg/kg. At this dose level, clinical signs consisted of increased incidences of dark faeces, diarrhoea and reduced faeces production. Food and water consumption was reduced. In addition, body weight gain was decreased, with several animals showing a transient body weight loss.
In animals treated with 75 mg/kg bw, dark faeces, diarrhea, reduced faeces production and slightly reduced food and water intake were also observed but because these changes were very limited and in view of the absence of more severe effects such as changes in body weight gain, these effects were not considered to be toxicologically relevant.
No maternal toxicity was observed in the 20 mg/kg dose group.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

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

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:
No treatment related effect on litter size was noted. The mean number of viable fetuses per litter was 9.9, 8.3, 8.8 and 8.3 for Groups 1, 2, 3 and 4,
respectively.

There were no treatment-related effects on the sex ratio of the fetuses.

No treatment related effect on fetal body weights were noted.

The mean fetal body weights (sexes combined) were 38.4, 41.4, 40.4 and 37.4 gram, respectively.

There were no treatment-related effects on fetal external morphology. External malformations were observed in fetus no. A024-09 at 20 mg/kg GLDA and in control fetus no. A006-08. The fetus in the low dose group had an omphalocele, whereby several loops of intestine and a portion of liver protruded through a defect in the abdominal wall at the umbilicus. The control fetus also had an omphalocele and in addition hyperextension of both hind limbs and absence of the anus and tail. There were no other fetuses with external malformations, and external developmental variations were not observed in this study.

There were no treatment-related effects on fetal visceral morphology. Visceral malformations were observed in 3(3), 5(5), 3(2) and 6(5) fetuses (litters) in the control, 20, 75 and 300 mg/kg groups, respectively. The most frequently observed malformation was absent lung lobe and in all cases, the accessory lung lobe was missing. In the control, 20, 75 and 300 mg/kg groups, respectively 1(1), 4(4), 2(2) and 5(4) fetuses (litters) were affected, resulting in mean litter incidences of respectively 0.6%, 2.2%, 1.0% and 3.2% per litter. The fetuses involved were nos. A010-09 (control group), A024-09, A033-02, A042-11, A043-08 (20 mg/kg group), A046-07, A057-04 (75 mg/kg group), A070-08, A072-03,05, A077-08 and A078-07 (300 mg/kg group). Of these fetuses, no. A024-09 also had an omphalocele externally, which was described in the paragraph above. Other visceral malformations in test substance-treated groups were observed in fetus no. A028-06 at 20 mg/kg (abnormal liver lobation), fetus no. A057-07 at 75 mg/kg (diaphragm cyst) and in fetus no. A068-01 at 300 mg/kg (atretic pulmonary trunk, dilated aortic arch and atrial septum defect). Besides, visceral malformations were observed in two control fetuses, namely in fetus no. A004-15 (persistent truncus arteriosus) and in fetus no. A006-08 (absent urinary bladder). The latter fetus also had multiple external malformations, which were described above. Visceral developmental variations observed in the test substance-treated groups were malpositioned left carotid, partially undescended thymus horn, small gallbladder, liver cyst, hemorrhagic thymus, retrocaval ureter, absent renal papilla, supernumerary artery, supernumerary spleen, small lung lobe, ovary cyst, bilobed gallbladder, liver appendix and discolored thymus. All but the last four findings were also observed in control fetuses.

There were no treatment-related effects on fetal skeletal morphology. Skeletal malformations were observed in 6(4) and 4(3) fetuses (litters) in the control and 300 mg/kg groups, respectively. Two different malformations were observed at 300 mg/kg. Three fetuses (nos. A085-01,02 and A087-01) from two litters had a vertebral anomaly with or without associated rib anomaly and one fetus (no. A088-01) had fused sternebrae. The same findings were also seen in
control fetuses, namely a vertebral anomaly in fetus no. A013-10 and fused sternebrae in fetus nos. A006-11, A020-01,05 and A022-07. Except for a case of spina bifida (that could not be detected externally) in control fetus no. A006-08, which also had malformations externally and viscerally, there were no other fetuses with skeletal malformations. Skeletal developmental variations observed in the high GLDA dose group were 13th full ribs, 13th rudimentary ribs, unossified sternebrae nos. 5 and/or 6, caudal shift of pelvic girdle, unossified line in skull bone, unossified metacarpals, 7th cervical rudimentary ribs, supernumerary sternal ossification sites, slightly to moderately malaligned sternebrae, wide sternebra, reduced ossification of vertebral centrum, supernumerary sternebra and 7th cervical full ribs. All but the last four findings were also observed in control fetuses.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on the results in this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) was established as 75 mg/kg body weight/day. The developmental NOAEL was at least 300 mg/kg body weight/day.

Executive summary:

The study was based on the following guidelines: 1) OECD 414, Prenatal Developmental Toxicity Study, January 2001. 2) EC No 440/2008 B.31: "Prenatal Developmental Toxicity Study", May 2008. 3) OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

Rationale for dose levels: Dose levels were based on a prenatal developmental toxicity study in New Zealand White (NZW) rabbits (Project 487520) in which doses of 30, 100 and 300 mg/kg were tested. No fetal malformations or developmental variations were noted in this study. As such the developmental NOAEL for GLDA was established to be at least 300 mg/kg. An increased number of non-pregnant females was, however, noted in all treated groups compared to the control group but these numbers were within the normal range or just below the historical control data and still considered normal. A dose response relationship was not present. Based on the reduced number of pregnancies the French Authorities, however, concluded that this study should be repeated with at least one dose level lower than 30 mg/kg. In consultation with the sponsor, dose levels of 20, 75 and 300 mg/kg were selected.

Study outline: Eighty-eight mated female NZW rabbits were assigned to four dose groups. The test item was administered once daily by gavage from Days 7 to 28 post-coitum at doses of 20, 75 and 300 mg/kg (Groups 2, 3 and 4 respectively). The rabbits of the control group received the vehicle, elix water, alone. Females were checked daily for the presence of clinical signs. Body weights and food consumption were determined at periodic intervals. Based on a possible treatment related effect, water consumption was determined daily from 12 June 2012 onwards. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies (all Groups) and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations (Groups 1 and 4).

Results: Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Maternal toxicity was noted at 300 mg/kg. At this dose level, clinical signs consisted of increased incidences of dark faeces, diarrhoea and reduced faeces production. Food and water consumption was reduced. In addition, body weight gain was decreased, with several animals showing a transient body weight loss. In animals treated with 75 mg/kg bw, dark faeces, diarrhea, reduced faeces production and slightly reduced food and water intake were also observed but because these changes were very limited and in view of the absence of more severe effects such as changes in body weight gain, these effects were not considered to be toxicologically relevant. No maternal toxicity was observed in the 20 mg/kg dose group. No developmental toxicity was observed in the 20, 75 and 300 mg/kg/day groups.

Conclusion: Based on the results in this prenatal developmental toxicity study, the maternal No Observed Effect Level (NOEL) for GLDA was established as 20 mg/kg body weight/day; the No Observed Adverse Effect Level (NOAEL) was established as 75 mg/kg body weight/day. The developmental NOAEL was at least 300 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Two rabbit studies are available for GLDA-Na4. One well performed study, and one study in which the health status of the animals was doubted because of a high number of non-pregnant animals. The absence of developmental effects in rabbits was supported by the results of the 2-generation study in rats in which additional examinations on the fetuses had been carried out.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In both rats and rabbits, there was no sign of (prenatal) developmental toxicity with GLDA-Na4. The NOAEL for developmental toxicity in rabbits (gavage study) was at least 300 mg/kg bw; the maternal NOAEL in rabbits was 75 mg/kg bw/day. The NOAEL for developmental toxicity and maternal toxicity in rats (dietary study) was at least 15,000 ppm. The latter level corresponded to 1230 -2668 mg GLDA-Na4 per kg bw for females.

Justification for selection of Effect on developmental toxicity: via oral route:
Well performed and reported study with the read across substance GLDA-Na4.

Justification for classification or non-classification

As no developmental/reproduction toxicity was observed at high levels of GLDA, no classification is needed for repro/developmental toxicity of GLDA-H4.