Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
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)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Appearance: white powder
Batch no.: CFC-8839 (see CoA in the report attached)
Content: 91.0 +/- 0.5 wt% Assay as GLDA-Na4
Storage: at room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 23 June 2010

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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

Administration / exposure

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)
Doses / concentrations
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

Examinations

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)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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

Effect levels (F1)

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

Results: F2 generation

Effect levels (F2)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.